Delivery of payloads to stem cells

ABSTRACT

The present disclosure relates to a method of targeting stems cells, in particular non-apoptotic stem cells, employing a GLA domain, capable of binding surface exposed phosphatidyl serine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/554,530 filed Sep. 5, 2017, U.S. Provisional Application No.62/554,533 filed Sep. 5, 2017, U.S. Provisional Application No.62/569,403 filed Oct. 6, 2017, U.S. Provisional Application No.62/569,411 filed Oct. 6, 2017, U.S. Provisional Application No.62/584,565 filed Nov. 10, 2017, and U.S. Provisional Application No.62/593,014 filed Nov. 30, 2017, each of which applications is hereinincorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

This application contains a sequence listing submitted electronicallyvia EFS-web, which serves as both the paper copy and the computerreadable form (CRF) and consists of a file entitled“ST-CT1-PCT_sequence.txt”, which was created on Sep. 5, 2018, which is9,831 bytes in size, and which is herein incorporated by reference inits entirety.

The present disclosure relates to a method of targeting stems cells, inparticular non-apoptotic stem cells, employing a GLA domain, for exampleto facilitate entry into the cell.

BACKGROUND

GLA domains are contained in a number of GLA proteins, such as Thrombin,Factor VII, Factor IX, Factor X, Protein C, Protein S (PrS), Protein Z,Osteocalcin, Matrix GLA protein, GAS6, Transthretin, Periostin, Prolinerich GLA 1, Proline rich GLA 2, Proline rich GLA 3 and Proline rich GLA4.

The GLA domains of so-called GLA proteins are able to bindphosphatidylserine (PtdS also referred to as PS) on the surface ofapoptotic cells, such as cancer cells and pathogen infected cells.Molecules excluding the catalytic domain, which specifically bindphosphatidyl serine are disclosed in WO2014/151535 and WO2014/151683,incorporated herein by reference.

GLA domains (Vitamin K-dependent carboxylation/gamma-carboxyglutamic)are protein domains which have been modified by vitamin K dependentpost-translational carboxylation of glutamate residues in the aminosequence to provide gamma-carboxyglutamate (Gla).

The GLA domain binds calcium ions by chelating them between twocarboxylic acid residues. These residues are part of a region thatstarts at the N-terminal extremity of the mature form of GLA proteins,and that ends with a conserved aromatic residue. This results in aconserved Gla-x(3)-Gla-x-Cys motif that is found in the middle of thedomain, which seems to be important for substrate recognition by thecarboxylase.

Phosphatidyl serine was thought to be a conserved marker for apoptoticcells and part of the mechanism by which diseased cells reduce immuneclearance or induced immune tolerance. Thus, it was hypothesized thatthe GLA domains only bound to apoptotic cells. However, surprisingly thepresent inventors have established that the GLA domains of the presentdisclosure can be employed to target stem cells, such as non-apoptoticstem cells and/or cancer stem cells. This is even more surprisingbecause the inventors have evidence to suggest that normally healthydifferentiated cells are not bound by the GLA domains employed in thepresent disclosure.

This has important implications, for example for stem therapy, which isused to treat conditions such as haematological cancers, such asleukemia. The stem cell therapy can only be given once the patient's ownbone marrow/stems cells/immune system has been wiped clean.

This wiping clean process requires “obliteration therapies”, for examplehigh doses of chemo, radiation therapy, and/or B cell depletion therapy.

This “obliteration therapy” has many side effects, for example mouth andthroat pain (which may make it difficult for the patient to eat), nauseaand vomiting, susceptibility to infection, such as pneumonia and CMVinfection, anemia, bleeding, infertility, cognitive dysfunction, etc.These side effects are very severe, and are difficult for patients,especially children to cope with. It would greatly improve patientquality of life if these side effects could be minimized or eliminated.

Wiping out the immune system and rebooting it has also been found to putaggressive forms of MS into the remission. However, the treatment isreserved for only the severest of cases because the risk associated withthe treatment are significant. However, the present disclosure allowsthe chemotherapy to be specifically targette to the stems by employingthe GLA-component.

The present invention provides a mechanism for “specifically” targetingstem cells, in particular non-apoptotic stem cells. Stem cells targetedby the method can, for example be isolated, treated (including geneticcorrection, augmentation, addition), labelled, transformed and/oreliminated. Thus, the method of the present disclosure can be employedto deliver therapeutics interventions to stem cells, for example geneticmaterial and/or proteineous material and/or chemical therapies.

By linking the GLA domain of the present disclosure to a detectablelabel, such as fluorescent label, his-tag or a magnetic bead, then stemscells can isolated and sorted etc. This may be useful in a diagnostic orisolating stems cells for further manipulation to render them useful intherapeutic application.

SUMMARY OF THE DISCLOSURE

The present disclosure will now be summarised in the “numbered”paragraphs below:

-   1a. A method of targeting a stem cell said method comprising the    step of contacting cells with a molecule comprising a payload linked    to a gamma-carboxyglutamic acid component (GLA-component) wherein    said GLA-component comprises a GLA domain or an active fragment    thereof, and does not comprise an active catalytic domain from a GLA    protein.-   1b. A molecule comprising a payload linked to a    gamma-carboxyglutamic acid component (GLA-component), wherein said    GLA-component comprises a GLA domain or an active fragment thereof,    and does not comprise an active catalytic domain from a GLA protein    for use in treatment or diagnosis of a stem cell-   1c. A molecule comprising a payload linked to a    gamma-carboxyglutamic acid component (GLA-component), wherein said    GLA-component comprises a GLA domain or an active fragment thereof,    and does not comprise an active catalytic domain from a GLA protein    for use in the manufacture of a medicament for treatment or    diagnosis of a stem cell.-   2. A method or molecule for use according to paragraph 1a, 1b or 1c,    wherein GLA domain or active fragment thereof is independently    selected from thrombin, factor VII, factor IX, factor X, protein C,    protein S, protein Z, osteocalcin, matrix GLA protein (MGP), GAS6,    transthyretin (TTR), inter-alpha-trypsin-inhibitor, periostin,    proline rich gla 1 (PRRG1), proline rich gla 2 (PRRG2), proline rich    gla 3(PRRG3), and proline rich gla 4 (PRRG4).-   3. A method or molecule for use according to paragraph 2, wherein    the GLA domain or active fragment thereof is independently selected    from thrombin, factor VII, factor IX, factor X, protein C, protein    S, protein Z and GAS6, for example the GLA domain from protein S, in    particular a sequence shown in SEQ ID NO: 1.-   4. A method or molecule for use according to paragraph 1a, 1b or 1c    to 3, wherein the GLA-component further comprises an EGF domain, for    example a calcium binding EGF domain.-   5. A method or molecule for use according to paragraph 1a, 1b or 1c    to 4, wherein the construct comprises an EGF domain selected from    thrombin, factor VII, factor IX, factor X, protein C, protein S,    protein Z, Osteocalcin, Matrix GLA protein, GAS6, Transthretin,    Periostin, Proline rich GLA 1, Proline rich GLA 2, Proline rich GLA    3 and Proline rich GLA 4.-   6. A method or molecule for use according to paragraph 5 wherein the    EGF domain selected from thrombin, factor VII, factor IX, factor X,    protein C, protein S, protein Z and GAS6, for example the EGF domain    from protein S-   7. A method or molecule for use according to any one of paragraphs    1a, 1b or 1c to 6, wherein the GLA-component comprises a sequence    shown in SEQ ID NO: 6 or a derivative thereof excluding the his-tag.-   8. A method or molecule for use according to paragraph 1a, 1b or 1c    to 7, wherein the GLA-domain component further comprises a Kringle    domain.-   9. A method or molecule for use according to paragraph 8, wherein    the Kringle domain is from a protein selected from the group    comprising Activating transcription factor 2 (ATF); Factor XII    (F12); thrombin (F2); Hyaluronan-binding protein 2 (HABP2);    Hepatocyte growth factor (HGF); Hepatocyte growth factor activator    (HGFAC); Kremen protein 1 (KREMEN1); KREMEN2; Lipoprotein(a) (LPA);    LPAL2; Macrophage-stimulating protein (MSP or MST1);    Phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1); Tissue    plasminogen activator (PLAT); Urokinase (PLAU); Plasmin (PLG);    PRSS12; Tyrosine-protein kinase transmembrane receptor ROR1 (ROR1);    and Tyrosine-protein kinase transmembrane receptor ROR2 (ROR2).-   10. A method according to any one of paragraphs 1a, 1b, 1c to 9,    wherein the method is performed in vitro.-   11. A method according to any one of paragraphs 1a, 1b, 1c to 10,    wherein the delivery is to a cell in vivo, for example wherein the    molecule comprising the GLA component and the payload are    administered to a patient, for example a human patient.-   12. A method or molecule for use according to paragraph 1a, 1b or 1c    to 11, wherein the molecule targets the exterior of a stem cell.-   13. A method or molecule for use according to paragraph 1a, 1b or 1c    to 12, wherein the molecule is internalized in a stem cell.-   14. A method or molecule for use according to paragraph 1a, 1b or 1c    to 13, wherein the cell is non-apoptotic (i.e. a healthy stem cell).-   15. A method or molecule for use according to paragraph 1a, 1b or 1c    to 13, wherein the cell is apoptotic, for example a diseased stem    cell.-   16. A method or molecule for use according to paragraph 1a, 1b or 1c    to 15, wherein the stem cell is an adult stems cell, for example    including progenitor cells, and haemotopoietic stem cells, myogenic    stem cells, osteoprogenitor stem cells, neural stem cells,    mesenchymal stem cell, such as satellite cells, radial glial cells,    bone marrow stromal cells, periosteum, pancreatic progenitor cells,    endothelial progenitor cells, blast cells and trophoblast stem    cells.-   17. A method or molecule for use according to paragraph 1a, 1b or 1c    to 16, wherein the stem cell expresses a surface marker CD34.-   18. A method or molecule for use according to paragraph 1a, 1b or 1c    to 17, wherein the stem cell is negative for lineage positive    surface markers (i.e. is Lin −ve).-   19. A method or molecule for use according to paragraph 1a, 1b or 1c    to 18, wherein the stem cell is Lin −ve, CD34 +ve, CD38 −ve, CD45RA    −ve, CD90 positive and CD49f +ve.-   20. A method or molecule for use according to paragraph 1a, 1b or 1c    to 17, wherein the stem cell is a haemotopoietic stem cell.-   21. A method or molecule for use according to paragraph 20, wherein    the stem cell expresses a surface marker from CD48, CD150, CD244,    CD34, CD38, SCA-1, Thy1.1, C-kit, lin, CD135, slam1/CD150, Mac-1    (CD11b), CD4, stem cell factor (SCF) and combinations of two or more    of the same.-   22. A method or molecule for use according to paragraph 1a, 1b or 1c    to 19, wherein the stem cell is an osteoprogenitor cell.-   23. A method or molecule for use according to paragraph 22, wherein    the stem cell expresses a surface marker selected from Gremlin-1,    TGF-beta, bFGF, BMP-2, ALPP, MCAM, Collagen I, Collagen 1 alpha 1,    Collagen II, RUNX2, Decorin, and combinations of two or more of the    same (such as all said markers).-   24. A method or molecule for use according to paragraphs 22 or 23,    wherein the stem cells are osteoblasts or a progenitor thereof.-   25. A method or molecule for use according to paragraph 24, wherein    the stem cell expresses a surface marker selected from Runx2,    alkaline phosphatase/ALPP/ALPI, osteocalcin, BAP1, OPN, BAP31,    Collagen I, SCUBE3, Fibronectin, SPARC, IGFBP-3, and combinations of    two or more of the same (such as all said markers).-   26. A method or molecule for use according to paragraph 1a, 1b or 1c    to 16, wherein the stem cell is an osteocyte or progenitor thereof.-   27. A method or molecule for use according to paragraph 26, wherein    the stem cell expresses a surface marker selected from TGF beta,    RANKL, MCSF, Sclerostin, DKK, and combinations of two or more of the    same (such as all said markers).-   28. A method or molecule for use according to paragraph 26, wherein    the stem cell expresses a surface marker selected from Osterix +ve,    CD90 +ve, osteocalcin +ve, collagen I +ve, bone sialoprotein +ve and    combinations of two or more of the same (such as all said markers).-   29. A method or molecule for use according to paragraph 26, wherein    the stem cell expresses a surface marker selected from alkaline    phosphatase/ALPP(alkaline phosphatase placental)/ALPI +ve, collagen    I +ve, collagen II +ve, decorin +ve, MCAM/CD146 +ve, MEPE/OF45 +ve,    osterix +ve, CD90 +ve, osterix/Sp7 +ve, RUNX2/CBFA1 +ve,    thrombopoietin/Tpo +ve, and combinations of two or more of the same    (such as all said markers).-   30. A method or molecule for use according to any one of paragraphs    1a, 1b or 1c to 19, wherein the stem cell is a myogenic stem cell.-   31. A method or molecule for use according to paragraph 30, wherein    the stem cell expresses a marker selected from CD56, CD146,    VE-cadherin, alpha-smooth muscle actin, FABP3, integrin alpha 7,    desmin, myosin heavy chain, UEA-1 receptor, and combinations of two    or more of the same (such as all said markers).-   32. A method or molecule for use according to paragraph 1a, 1b or 1c    to 19, wherein the stem cell is a neural stem cell.-   33. A method or molecule for use according to paragraph 32, wherein    the stem cell expresses a marker selected from CD133, CD15, CD24 low    or −ve, GCTM-2, CD45, CD34, Nestin, Sox-2, ABCG2, FGF R4,    Frizzled-9, and combinations of two or more of the same (such as all    said markers).-   34. A method or molecule for use according to paragraph 33, wherein    the CD24 marker is low or −ve.-   35. A method or molecule for use according to paragraphs 33 or 34,    wherein the wherein the stem cell expresses a marker combination of    CD133 +ve, 5E12 +ve, CD34 −ve, CD45 −ve, and CD24 low or −ve.-   36. A method or molecule for use according to paragraph 1a, 1b or 1c    to 19, wherein the stem cell is a mesenchymal stem cell.-   37. A method or molecule for use according to paragraph 36, wherein    the stem cell expresses a surface marker, selected from CD10, CD13,    CD73, CD105, CD271, CD140b, CD240, frizzled-9, CD29, CD90, CD146,    oct4, SSEA4, STRO-1, stem cell factor (SCF) and combinations of two    or more of the same.-   38. A method or molecule for use according to paragraph 1a, 1b or 1c    to 19, wherein the stem cell is adipose-derived.-   39. A method or molecule for use according to paragraph 38, wherein    the stem cell expresses a surface marker selected from K15, CD34,    Nestlin, follistatin, p63, integrin alpha 6, teacin C, EGFR, IGFR,    frizzled factors, and combinations of two or more of the same.-   40. A method or molecule for use according to paragraphs 38 or 39,    wherein the stem cell expresses a surface marker selected from CD44,    ICAM/CD54, CD34, integrin family members and combinations of two or    more of the same.-   41. A method or molecule for use according to paragraph 1a, 1b or 1c    to 19, wherein stem cell is an ovary and tubal epithelial stem cell.-   42. A method according to paragraph 41, wherein the stem cell    expresses a surface marker selected from Gremlin 1, Lrig1, Lgr5,    Bmi1, Tert, HopX and combinations of two or more of the same.-   43. A method or molecule for use according to paragraph 1a, 1b or 1c    to 15, wherein the stem cell is an embryonic stem cell.-   44. A method or molecule for use according to paragraph 43, wherein    the stem cell can be identified on the basis of one or more a    surface marker selected from CD24, CD29, CD31, CD59, CD90, CD117,    CD133, CD324, CD326, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, frizzled5,    stem cell factor, crypto (TDGF-1)-   45. A method or molecule for use according to paragraph 1a, 1b or 1c    to 44, wherein the stem cell is non-cancerous.-   46. A method or molecule for use according to paragraph 1a, 1b or 1c    to 44, wherein the stem cell is a cancer stem cell.-   47. A method or molecule for use according to paragraph 46, wherein    the cancer stem cell is of epithelial origin.-   48. A method or molecule for use according to paragraph 46 or 47,    wherein cancer stem cell expresses a surface marker selected from    CD44 (which is over expressed in at least breast, ovary, prostate,    pancreatic, squamous, and bladder cancer), CD133 (which is over    expressed in at least brain, colon, lung, prostate cancer and    medulloblastoma), CD24, CD90, CD271, CD4f, CD13 and combinations of    two or more of the same (other markers include ABCB5⁺, CD44+/CD24,    CD34⁺/CD38⁻, and CD44⁺/ESA⁺).-   49. A method or molecule for use according to paragraph 46, wherein    the stem cell is of haemotopoietic origin.-   50. A method or molecule for use according to paragraph 49, wherein    the cell has a marker selected from CD19, WT-1 and combinations    thereof-   51. A method or molecule for use according to any one of paragraphs    1 to 50 wherein the GLA-component is conjugated to the payload.-   52. A method or molecule for use according to paragraph 1a, 1b or 1c    to 51, wherein the payload comprises a therapeutic agent, a    targeting agent and/or a label.-   53. A method or molecule for use according to paragraph 52, wherein    the therapeutic agent is a chemical entity, or biological molecule    (such as a protein), for example, an anti-cancer drug, an    anti-cancer therapy, a chemotherapeutic agent, virus or viral    vector, such as an oncolytic virus.-   54. A method or molecule for use according to paragraph 1a, 1b or 1c    to 53, wherein the payload is selected from a toxin, a polymer(for    example synthetic or naturally occurring polymers), biologically    active proteins (for example enzymes, other antibody or antibody    fragments), a drug (for example, small molecule (chemical entity) or    chemotherapeutic agent), nucleic acids and fragments thereof (for    example DNA, RNA such as shRNA and siRNA and fragments thereof,    including gRNA for CRISPRCas9 and CRISPRa/i, radionuclides    (particularly radioiodide, radioisotopes) a metal chelating agent,    nanoparticles and reporter groups (such as fluorescent or    luminescent labels or compounds which may be detected by NMR or ESR    spectroscopy).-   55. A method or molecule for use according to paragraph 54, wherein    the toxin is selected from an auristatin (for example MMAE    (monomethyl auristatin E), MMAF (monomethyl auristatin F)),    pyrrolobenzodiazepine (PBD), doxorubicin, duocarmycin, a    maytansinoid (for example N 2′-deacetyl-N    2′-(3-mercapto-1-oxopropyl)-maytansine (DM1), N    2′-deacetyl-N2′-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N    2′-deacetyl-N 2′(4-methyl-4-mercapto-1-oxopentyl)-maytansine (DM4)),    calocheamicin, dolastatin, maytansine, α-amanitin, Pseudomonas    exotoxin (PE38), ricin A chain, diphtheria toxin, Pokeweed antiviral    protein (PAP), saporin, gelonin and a tubulysin.-   56. A method or molecule for use according paragraph 53, wherein the    chemotherapeutic is selected from temozolomide, epothilones,    melphalan, carmustine, busulfan, lomustine, cyclophosphamide,    dacarbazine, polifeprosan, ifosfamide, chlorambucil,    mechlorethamine, busulfan, cyclophosphamide, carboplatin, cisplatin,    thiotepa, capecitabine, streptozocin, bicalutamide, flutamide,    nilutamide, leuprolide acetate, doxorubicin hydrochloride, bleomycin    sulfate, daunorubicin hydrochloride, dactinomycin, liposomal    daunorubicin citrate, liposomal doxorubicin hydrochloride,    epirubicin hydrochloride, idarubicin hydrochloride, mitomycin,    doxorubicin, valrubicin, anastrozole, toremifene citrate,    cytarabine, fluorouracil, fludarabine, floxuridine, interferon α-2b,    plicamycin, mercaptopurine, methotrexate, interferon α-2a,    medroxyprogersterone acetate, estramustine phosphate sodium,    estradiol, leuprolide acetate, megestrol acetate, octreotide    acetate, deithylstilbestrol diphosphate, testolactone, goserelin    acetate, etoposide phosphate, vincristine sulfate, etoposide,    vinblastine, etoposide, vincristine sulfate, teniposide,    trastuzumab, gemtuzumab ozogamicin, rituximab, exemestane,    irinotecan hydrocholride, asparaginase, gemcitabine hydrochloride,    altretamine, topotecan hydrochloride, hydroxyurea, cladribine,    mitotane, procarbazine hydrochloride, vinorelbine tartrate,    pentrostatin sodium, mitoxantrone, pegaspargase, denileukin    diftitix, altretinoin, porfimer, bexarotene, paclitaxel, docetaxel,    arsenic trioxide, tretinoin and combinations of two or more of the    same.-   57. A method or molecule for use according to any one of paragraphs    52 to 54, wherein the chemotherapeutic is selected from an    alkylating agent, an antimetabolite including thymidylate synthase    inhibitors, a taxane, an anthracycline, an anti-microtubule agent    including plant alkaloids, and combinations of two or more of the    same.-   58. A method or molecule for use according to paragraph 57, wherein    the chemotherapeutic is selected from paclitaxel, docetaxel,    abraxane, carbazitaxel, derivatives of any one of the same, and    combinations of two or more of any of the aforementioned.-   59. A method according to paragraph 57 or 58, wherein the alkylating    agent is selected from a nitrogen mustard, a nitrosourea (such as    carmustine), a tetrazine, a aziridine, a platin and derivatives    thereof, a non-classical alkylating agent and a combination of two    or more of the same.-   60. A method or molecule for use according to paragraph 59, where    the platin is selected from cisplatin, carboplatin, oxaliplatin,    satraplatin, picoplatin, nedaplatin, triplatin, lipoplatin and a    combination of two or more of the same. 61. A method or molecule for    use according to any one of paragraphs 57 or 58, wherein the    alkylating agent is an antimetabolite selected from anti-folates    (for example methotrexate and pemetrexed), purine analogues (for    example thiopurines, such as azathiopurine, mercaptopurine,    thiopurine, fludarabine (including the phosphate form), pentostatin    and cladribine), pyrimidine analogues (for example    fluoropyrimidines, such as 5-fluorouracil and prodrugs thereof such    as capecitabine [Xeloda®]), floxuridine, gemcitabine, cytarabine,    decitabine, raltitrexed(tomudex) hydrochloride, cladribine and    6-azauracil and combination of two or more thereof.-   62. A method or molecule for use according to any one of paragraph    56 to 60, wherein the anthracycline is selected from daunorubicin    (Daunomycin), daunorubicin (liposomal), doxorubicin (Adriamycin),    doxorubicin (liposomal), epirubicin, idarubicin, mitoxantrone and a    combination of two or more thereof, in particular doxorubicin.-   63. A method or molecule for use according to any one of paragraphs    53 to 62, wherein the drug is an anti-cancer drug, for example    selected from a topoisomerase inhibitor, a PARP inhibitor and a    combination of or more of the same.-   64. A method or molecule for use according to any one of paragraph    53 to 64, wherein the anti-cancer therapy is a radionuclide, for    example selected from Y-90, P-32, I-131, In-111, Sr-89, Re-186,    Sm-153, Sn-117m and a combination of two or more of the same.-   65. A method or molecule for use according to any one of paragraphs    1a, 1 b or 1c to 64 which comprises administering the molecule    comprising the GLA component and payload to a cancer patient, for    example where the cancer is refractory.-   66. A method or molecule for use according to paragraph 65, wherein    the cancer is an epithelial cancer, for example colorectal cancer,    testicular cancer, liver cancer, biliary tract cancer,    glioblastomer, melanoma, prostate cancer, pancreatic cancer, breast    cancer, ovarian cancer, cervical cancer, uterine cancer, gastric    cancer, oesophageal cancer, thyroid cancer, renal cancer, bladder    cancer, brain cancer, head and neck cancer or lung cancer or    alternatively the cancer may be a haematological cancer, for example    leukaemia, lymphoma, myeloma and chronic myeloproliferative    diseases, such as AML, CML, ALL and CLL.-   67. A method or molecule for use according to any one of paragraphs    1 to 66, wherein the payload is converted to an active form inside    the cell.-   68. A method or molecule for use according to paragraph 67, wherein    conversion to the active form is performed by an enzyme.-   69. A method or molecule for use according to paragraph 68, wherein    the enzyme is selected from a carboxylesterase,    acetylecholinesterase, paraoxonase (such as paraoxonase 2), matrix    metalloproteases, alkaline phosphatase, β-glucuronidase,    purine-nucleoside phosphorylase, beta-lactamase (for example    produced by Mycobacterium tuberculosis and kansasii) and cytosine    deminase.-   70. A method or molecule according to any one of claims 1 to 69,    wherein the GLA-component has the sequence shown in SEQ ID NO: 6 or    an equivalent sequence excluding the His-tag.

In one embodiment the GLA-component binds surface exposedphosphatidylserine on the cells, before internalisation.

Whilst not wishing to be bound by theory the present inventors believethat not all phosphatidylserine is equivalent from a biologicalperspective. The inventors believe that the phosphatidylserine exposesby the enzyme TMEM16F is involved in immune suppression and is the one“seen” by the molecules of the present disclosure.

In one embodiment the stem cell is an adult stem cells or vesiclederived therefrom, for example a somatic stem cells, such as ahematopoetic stem cell, a mesenchymal stem cell, or a stromal stem cell.

In one embodiment the stem cell is an embryonic stem cell or a vesiclederived therefrom. In one embodiment the cell is not an embryonic stemcell.

In one embodiment the method relates to mammalian stem cells, forexample human stem cells. The stem cell discussed herein are primarilyhuman stem cells. However, the skilled person is able to identify therelevant or corresponding stem cell population for other mammals, asrequired. For example SSEA-1 is a marker for murine embryonic stemcells, human germline cells and embryonal carcinoma cells; SSEA-3 is amarker for primate embryonic stem cells, human embryonic germline cells,human embryonic stem cells and embryonal carcinoma cells; SSEA-4 is amarker for primate embryonic stem cells, human embryonic germ cells,human stem cells, embryonal carcinoma cells; CD324 is a marker for human& murine embryonic stem cells, embryonal cancer cells; CD90 is a markerfor human & murine embryonic stems cells, hematopoietic stem cells,embryonal carcinoma cells; CD117 is a marker for human & murineembryonic stem cells, hematopoietic stem progenitor cells, neuralcrest-derived melanocytes, primordial germ cells, embryonal carcinomacells; CD326 is a marker for human & murine embryonic stem cells,embryonal carcinoma cells; CD9 is a marker for human & murine embryonicstems; CD24 is a marker for human & murine embryonic stems; CD29 is amarker for human & murine embryonic stems; CD59 is a marker for human &murine embryonic stems; CD133 is a marker for human & murine embryonicstems, embryonal carcinoma cells, hematopoietic stem cells; CD31 is amarker for human & murine embryonic stems; TRA-1-60 is a marker forhuman embryonic stem cells, teracarcinoma, embryonic germ cells,embryonal carcinoma cells; TRA-1-81 is a marker for human embryonic stemcells, teracarcinoma, embryonic germ cells, embryonal carcinoma cells;Frizzled5 is a marker for human & murine embryonic stem cells; Stem cellfactor (SCF) is a marker for human & embryonic stem cells, hematopoieticstem cells, mesenchymal stem cells, embryonal carcinoma cells; andCripto is a marker for human & murine embryonic stem cells,cardiomyocytes and embryonal carcinoma cells.

In one embodiment the payload comprises a therapeutic agent.

In one embodiment the payload comprises a detectable label.

In one embodiment the payload comprises a DNA or RNA sequence, forexample cDNA comprising a transgene or an RNAi sequence (such as miRNA,siRNA including shRNA). The DNA encoding a transgene may be delivered atranscriptionally active DNA or a plasmid for transient or stableexpression.

In one embodiment the payload is suitable for inducing differentiationof the stem cell, for example to activate and/or mature the cell into aspecific lineage.

In one embodiment the method of the present disclosure comprises apre-treatment of a patient, for example to induce or augment expressionof PS on stem cells, for example the pre-treatment step may be treatmentwith radiation therapy, in particular irradiation of bone marrow cells.

The disclosure also extends to pharmaceutical compositions comprising amolecule for use according to the present disclosure, in particular foruse as described herein. Thus, in one embodiment the molecules accordingto the present disclosure are employed in the treatment of anintra-cellular target.

The present disclosure also extends to the use of a GLA-componentcomprises a GLA domain or an active fragment thereof, wherein saidGLA-component does not comprise an active catalytic domain from a GLAprotein, for intracellular targeting and delivery (includingintracellular delivery of the payload).

The present disclosure also extends to the use of a GLA-componentcomprises a GLA domain or an active fragment thereof, wherein saidGLA-component does not comprise an active catalytic domain from a GLAprotein, for the manufacture of a medicament for intracellular targetingand delivery (including intracellular delivery of the payload, inparticular where the payload comprises a therapeutic entity/molecule).

The present technology may be used to wipe out the immune cells ofpatient before stem cells transplantation, for example the payload willgenerally be a chemotherapy, for example comprising carmustine.

A current regime for immune cell ablation is (BCNU) 300 mg/m² on day −6,etoposide 200 mg/m² and cytarabine 200 mg/m² daily from day −5 to −2,and melphalan 140 mg/m² on day −1 (BEAM). Rabbit antithymocyte globulin(2.5 mg/kg/d) was administered on days −2 and −1. This regime can beadapted by conjugating each of the agents to a GLA molecule of thepresent disclosure.

In one embodiment the immune obliteration is for cancer, for ahematological cancer (for example is selected from myeloma, lymphoma,leukaemia, such as acute myeloid leukaemia (AML), chronicmyeloproliferative disease, monoclonal gammopathy of uncertainsignificance, myelodysplastic syndrome and amyloidosis, such as AML,CML, CLL or ALL)

In one embodiment the myeloma is selected from multiple myeloma,amyloidosis and plasmacytoma.

In one embodiment the myeloma is selected from monoclonal gammopathy ofundetermined significance, asymoptomatic myeloman, symptomatic myelomaand Kahler's disease.

In one embodiment the lymphoma is selected from anaplastic large celllymphoma, Burkitt lymphoma, Burkitt-like lymphoma, cutaneous T-celllymphoma, diffuse large B-cell lymphoma, diffuse large B-cell lymphoma,lymphoblastic lymphoma, MALT lymphoma, mantle cell lymphoma, mediastinallarge B-cell lymphoma, nodal marginal zone B-cell lymphoma, smalllymphocytic lymphoma, thyroid lymphoma, and Waldenstrom'smacroglobulinaemia.

In one embodiment the chronic myeloproliferative disease is selectedfrom essential thrombocythaemia, chronic idiopathic myelofibrosis, andpolycythaemia rubra vera.

In one embodiment the leukaemia is selected from acute myeloid leukaemia(AML), hairy cell leukaemia, acute lymphoblastic leukaemia, and chroniclymphoblastic leukaemia, such as AML.

It has recently become apparent that for severe autoimmune diseases,such as severe multiple sclerosis and severe arthritis obliteration ofimmune cells followed by stem transplant can put the disease intoremission. Thus, the ablation therapy of the present disclosure may beemployed in an autoimmune disease, such as multiple sclerosis andarthritis.

In one embodiment the GLA molecule of the present disclosure is linked,for example conjugated, to a payload which comprises a detectable label.Examples of detectable labels are given below. The detectable label canbe employed to sort or isolate the stem cells, for example employingFACs sorting, magnetic sorting or similar. Thus, in one embodiment thereis provided a method isolating or enriching stem cells employing aGLA-molecule of the present disclosure. This is advantages becausehistorically the isolation of the certain stem cell populations, such ascancer stem cells has been very difficult.

The labelled GLA molecule may also be employed in vivo as an imagingagent, in particular as a diagnostic tool, for example to identifycancer stem cells in primary tumors or metastasise. This may beimportant for monitoring patients after surgery and/or chemotherapy toensure the cancer is in remission.

The DNA transgene payloads and/or RNA payloads linker to the GLAmolecule can be employed as an alternative intracellular delivery to aviral vector delivery (transduction) or traditional transfection. Thiscan be employed to in vitro to express exogenous or endogenous proteinsin the cell (for example where the modified stem cells are forreinfusion into a patient) or can be effected in vivo. The genes can beexpressed transiently or can be designed to be stably integrated intothe stem cell.

Surprisingly the present inventors have shown that the moleculesaccording to the present disclosure not only bind stem cells they arerapidly internalised therein along with the payload attached thereto.

DETAILED DISCLOSURE

Intra-cellular delivery as employed herein refers conveying, for examplethe payload to inside the cell.

In one embodiment the payload is not internalized.

In one embodiment the GLA-component and the payload is not internalised.

Payload as employed herein refers to a molecule which is linked to theGLA domain, in particular for the purpose of intracellular delivery. Thelink may be a link through chemical conjugation using, for examplemaleimide chemistry or click chemistry to anchor to moiety to a solventexposed lysine. Alternatively, the link may be a fusion, for example apeptide bond where the linked entity is expressed as a fusion proteinwith the GLA component, for example this may be suitable for certaindetectable labels, such as fluorescent proteins or antibodies. Linkersmay be employed between the GLA-component and the payload. Payloads maycomprising a drug, a toxin, a polymer, a biologically active protein,therapeutic virus, oncolytic virus, viral vector, radionuclides, a metalchelating agent and/or a reporter group (such as a label).

In one embodiment 1, 2, 3, 4 or 5 payloads are linked per GLA-component.

GLA-component (also referred to herein as a gamma-carboxyglutamic acidcomponent) refers to a polypeptide comprising a GLA-domain in theabsence of catalytic domain from a GLA protein, such as protein S. Thepolypeptide may further comprise an EGF domain and/kringle domain, forexample from protein S. In one embodiment the GLA-component comprises 30to 300 amino acid residues, for example 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250,260, 270, 280, 290 or 300 residues. In one embodiment the GLA componentis in the range of 4.5 to 30 kDa. In one embodiment the GLA-componentcomprises the sequence shown in SEQ ID NO: 1. In one embodiment theGLA-component comprises a sequence shown in SEQ ID NO: 6 or a derivativethereof excluding the his-tag.

GLA domains (Vitamin K-dependent carboxylation/gamma-carboxyglutamic) asemployed herein are protein domains which have been modified by vitaminK dependent post-translational carboxylation of glutamate residues inthe amino sequence to provide gamma-carboxyglutamate (Gla). In oneembodiment the GLA domain employed in the molecules of the presentdisclosure comprises 30 to 45 consecutive residues from a native(wild-type) GLA domain. In one embodiment the GLA domain comprises 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 GLA residues.

In one embodiment 30% or less of the GLA-component is GLA residues.

In one embodiment the GLA-component comprises 1 to 5 disulfide bonds,for example 1, 2, 3, 4 or 5 disulfide bonds.

The GLA domain binds calcium ions by chelating them between twocarboxylic acid residues. These residues are part of a region thatstarts at the N-terminal extremity of the mature form of Gla proteins,and that ends with a conserved aromatic residue. This results in aconserved Gla-x(3)-Gla-x-Cys motif that is found in the middle of thedomain, and which seems to be important for substrate recognition by thecarboxylase.

GLA domains are contained in a number of proteins, such as Thrombin,Factor VII, Factor IX, Factor X, Protein C, Protein S (PrS), Protein Z,Osteocalcin, Matrix GLA protein, GAS6, Transthretin, Periostin, Prolinerich GLA 1, Proline rich GLA 2, Proline rich GLA 3, and Proline rich GLA4.

GLA domain as employed herein also extends to proteins where 1 to 10percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids inthe native GLA domain may be replaced and deleted, provided thatmodified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native(unmodified GLA domain) in a suitable in vitro assay.

EGF domain as employed herein refers is a conserved protein domain. Itcomprises about 30 to 40 amino-acid residues and has been found in alarge number of mostly animal proteins. Most occurrences of the EGF-likedomain are found in the extracellular domain of membrane-bound proteinsor in proteins known to be secreted. The EGF-like domain includes 6cysteine residues. The main structure of EGF-like domains is atwo-stranded β-sheet followed by a loop to a short C-terminal,two-stranded β-sheet. These two β-sheets are usually denoted as themajor (N-terminal) and minor (C-terminal) sheets. EGF-like domainsfrequently occur in numerous tandem copies in proteins: these repeatstypically fold together to form a single, linear solenoid domain blockas a functional unit. In one embodiment the domain employed is thefull-length native domain.

EGF domain as employed herein also extends to proteins where 1 to 10percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids inthe native EGF domain may be replaced and deleted, provided thatmodified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native(unmodified EGF domain) in a suitable in vitro assay. In one embodimentthe protein is the full-length native domain.

Kringle domain as employed herein refers to autonomous protein domainsthat fold into large loops stabilized by 3 disulfide bonds. They arecharacterized by a triple loop, 3-disulfide bridge structure, whoseconformation is defined by a number of hydrogen bonds and small piecesof anti-parallel beta-sheet. They are found throughout the bloodclotting and fibrinolytic proteins, in a varying number of copies, insome plasma proteins including prothrombin and urokinase-typeplasminogen activator, which are serine proteases belonging to MEROPSpeptidase family S1A.

Kringle domain as employed herein also extends to proteins where 1 to 10percent (such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%) of the amino acids inthe native kringle domain may be replaced and deleted, provided thatmodified domain retains at least 70% (such as 75, 80, 85, 90, 91, 92,93, 94, 95, 96, 97, 98, 99 or 100%) of the native activity of the native(unmodified Kringle domain) in a suitable in vitro assay. In oneembodiment the domain employed is the full-length native domain.

An active fragment of a protein as employed herein is a less than thewhole native protein (or relevant domain), which retains at least 50%(such as 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%)of the active of the native full-length domain or protein in a relevantin vitro assay.

Catalytic domain as employed herein is a domain (or fragment) downstreamof the EGF domain in the C-terminal direction, for example asillustrated in FIG. 1A.

In vitro as employed herein refers to laboratory work not performed in ahuman or animal body.

In vivo as employed herein refer to work/testing/treatment in a livingorganism, in particular a human or animal.

Stem cell as employed herein refers to undifferentiated cells that arecapable of differentiation and includes embryonic stem cells and adultstem cells, in particular adult stem cells.

Hematopoietic stem cells (HSCs) or hemocytoblasts are the stem cellsthat give rise to all the other blood cells through the process ofhaematopoiesis. They are derived from mesoderm and located in the redbone marrow, which is contained in the core of most bones.

Cancer stem cell as employed herein refers to tumorigenic cells (i.e.cancer cells found within tumors or hematological cancers) that possesscharacteristics associated with normal stem cells, specifically theability to give rise to all cell types found in a particular cancersample. See, for example Identification and Targeting of Cancer StemCells, BioessayS 2009 October; 31 (10) 1038-1049. Cancer stem cells aredefined by three distinct properties: i) a selective capacity toinitiate tumour and drive neoplastic proliferation: ii) an ability tocreate endless copies of themselves through self-renewal, and iii) thepotential to give rise to more mature non-stem cell cancer progenythough differentiation. Cancer stem cells are not necessarily derivedfrom a healthy stem cell but may originate from a differentiated cell.

CD34 is also known as hematopoietic progenitor cells antigen CD34, has afunction as cell-cell adhesion factor. It can be employed as a marker toenrich stem populations.

Molecule as employed herein is used in the broadest sense and includes asynthetic chemical molecule but also macromolecules such as proteins,polymers (natural or otherwise), ribonucleic acid molecules, labels etc.

Payloads may comprising a drug, a toxin, a polymer, a biologicallyactive protein, radionuclides, a metal chelating agent and/or a reportergroup.

A drug as employed herein, unless the context indicates otherwise, isintended to refer to a small chemical entity, for example which has beensynthesised by organic chemistry methods, in particular a moleculeapproved or licensed or in the process of being licensed for therapeuticuse, especially in humans. Drug as employed herein also includes ananti-viral compound, an antibiotic, and an anti-cancer therapy.

An antiviral compound (antiviral agent) as employed herein refers to theclass of medicaments used specifically for treating viral infections,including broad spectrum anti-viral agents and also “narrow” spectrumspecific to a particular virus or particular family of viruses.

Antibiotic as employed herein refers to medicine or agent that inhibitsthe growth of bacteria or destroys bacteria. Anti-bacterial andantibiotic are used interchangeable here unless the context indicatesotherwise.

Anti-parasitic as employed herein in refers to a medicine or agent thatinhibits the growth of parasite, destroys parasite or removes parasitesfrom the host.

Anti-cancer therapy is a broad term which includes anti-cancer drugs,chemotherapy, radiotherapy, immune-oncology therapies, etc.

Anti-cancer drug as employed herein generally refers to a small moleculecancer therapy.

Chemotherapy as herein generally refers to a cytotoxic agent andincludes antineoplastics.

A biological therapeutic (also referred to as a biopharmaceutical,biological or biologic) is a therapeutic product “derived” from abiological source, for example a recombinant proteins and fragments,including antibodies molecules, including antibodies, antibody bindingfragments and multispecific antibody molecules and complex combinationsof such materials. A biologically active protein is a subgroup of abiological therapeutics and includes recombinant proteins and activefragments thereof (including antibody molecules).

Antibody molecules as employed herein include a complete antibody havingfull length heavy and light chains or a fragment thereof and a moleculecomprising any one of the same for example a Fab, modified Fab, Fab′,modified Fab′, F(ab′)2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies(e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies,Bis-scFv, diabodies, triabodies, tetrabodies and epitope-bindingfragments of any of the above (see for example Holliger and Hudson,2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, DrugDesign Reviews—Online 2(3), 209-217). The methods for creating andmanufacturing these antibody fragments are well known in the art (seefor example Verma et al., 1998, Journal of Immunological Methods, 216,165-181). Other antibody fragments for use in the present inventioninclude the Fab and Fab′ fragments described in International patentapplications WO2005/003169, WO2005/003170 and WO2005/003171.Multi-valent antibodies may comprise multiple specificities e.gbispecific or may be monospecific (see for example WO 92/22853 andWO05/113605). Bispecific and multispecific antibody variants areespecially considered in this example since the aim is to neutralise twoindependent target proteins. Variable regions from antibodies disclosedherein may be configured in such a way as to produce a single antibodyvariant which is capable of binding to and neutralising two targetantigens.

Antibody and binding fragments thereof, in particular small antibodyfragments such as domain antibodies, VHHs, single chain Fvs (scFvs),ds-scFvs and dsFv, may be delivered intracellularly using the presenttechnology.

In one embodiment the antibody or binding fragment thereof is acheckpoint inhibitor, for example an anti-PD-1 or anti-PD-L1 inhibitor.

In one embodiment the antibody molecule is human or humanised.

A toxin is a poisonous substance, especially derived from a naturalsource, in particular a protein. Many toxins, such as calicheamicin areused in cancer therapy. In addition chemotherapeutic agents can beconsidered toxic (or toxins). Thus the definition of toxin overlaps withother definitions herein. However, neurotoxins like snake venom aretoxin but not a chemotherapeutic. However, those skilled in the art arefamiliar with these technical definitions and are capable ofunderstanding the meaning the context of the present disclosure.

Diagnostic as employed herein is agent used in analysis or imaging todiagnose, label or monitor or understand a disease status. A diagnosticwill generally comprise a reporter molecule, such as a label or similarthat can visualised, measured or monitored in some way.

Radionuclides suitable for use the present disclosure includethallium-201, technetium-99m, Iodine-123, Iodine 131, Iodine-125,Fluorine-18 and Oxygen-15.

Also, of particular interest, is using GLA-component to deliverintrabodies, for example via GLA-fusions, for example where theintrabody is fused to the N or C terminus of the GLA-component.Intrabodies are able to target intracellular antigens.

In one embodiment antibodies that interact and inhibit RAS or proteinsin the RAS signaling pathway are employed in the payload. RAS genesconstitute a multigene family that includes HRAS, NRAS, and KRAS. RASproteins are small guanosine nucleotide-bound GTPases that function as acritical signaling hub within the cell. The RAS/MAPK pathway has beenstudied extensively in the context of oncogenesis because its somaticdysregulation is one of the primary causes of cancer. RAS is somaticallymutated in approximately 20% of malignancies (Bos J L, Cancer Res. 49:4682-4689, 1989). In this particular case, it is envisioned that, forexample the GLA-component is fuses to a RAS intrabody (described inCetin M et al., J Mol Biol. 429:562-573, 2017).

Apoptosis as employed herein is cell death pathway which occurs asnormal and controlled part an organism growth. Cell death by apoptosisis less damaging to surrounding tissue than cell death mechanisms, suchas necrosis.

Necrosis as employed herein is cell death from disease or injury. Itreleases cytokines and factors into the surrounding tissue that maydamage surrounding cells. Gangrene is an example of necrotic cell death.

Chemotherapeutic Agents

Chemotherapeutic agent and chemotherapy or cytotoxic agent are employedinterchangeably herein unless the context indicates otherwise.

Chemotherapy as employed herein is intended to refer to specificantineoplastic chemical agents or drugs that are “selectively”destructive to malignant cells and tissues, for example alkylatingagents, antimetabolites including thymidylate synthase inhibitors,anthracyclines, anti-microtubule agents including plant alkaloids,topoisomerase inhibitors, parp inhibitors and other antitumour agents.Selectively in this context is used loosely because of course many ofthese agents have serious side effects.

The preferred dose may be chosen by the practitioner, based on thenature of the cancer being treated.

Examples of alkylating agents, which may be employed in the method ofthe present disclosure include an alkylating agent nitrogen mustards,nitrosoureas, tetrazines, aziridines, platins and derivatives, andnon-classical alkylating agents.

Example a platinum containing chemotherapeutic agent (also referred toas platins), such as cisplatin, carboplatin, oxaliplatin, satraplatin,picoplatin, nedaplatin, triplatin and lipoplatin (a liposomal version ofcisplatin), in particular cisplatin, carboplatin and oxaliplatin.

The dose for cisplatin ranges from about 20 to about 270 mg/m² dependingon the exact cancer. Often the dose is in the range about 70 to about100 mg/m².

Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan,chlorambucil, ifosfamide and busulfan.

Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU),lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin.Tetrazines include dacarbazine, mitozolomide and temozolomide.

Aziridines include thiotepa, mytomycin and diaziquone (AZQ). Examples ofantimetabolites, which may be employed in the method of the presentdisclosure, include anti-folates (for example methotrexate andpemetrexed), purine analogues (for example thiopurines, such asazathiopurine, mercaptopurine, thiopurine, fludarabine (including thephosphate form), pentostatin and cladribine), pyrimidine analogues (forexample fluoropyrimidines, such as 5-fluorouracil and prodrugs thereofsuch as capecitabine [Xeloda®]), floxuridine, gemcitabine, cytarabine,decitabine, raltitrexed(tomudex) hydrochloride, cladribine and6-azauracil.

Examples of anthracyclines, which may be employed in the method of thepresent disclosure, include daunorubicin (Daunomycin), daunorubicin(liposomal), doxorubicin (Adriamycin), doxorubicin (liposomal),epirubicin, idarubicin, valrubicin currenity used only to treat bladdercancer and mitoxantrone an anthracycline analog, in particulardoxorubicin.

Examples of anti-microtubule agents, which may be employed in the methodof the present disclosure, include include vinca alkaloids and taxanes.

Vinca alkaloids include completely natural chemicals for examplevincristine and vinblastine and also semi-synthetic vinca alkaloids, forexample vinorelbine, vindesine, and vinflunine

Taxanes include paclitaxel, docetaxel, abraxane, carbazitaxel andderivatives of thereof. Derivatives of taxanes as employed hereinincludes reformulations of taxanes like taxol, for example in amicelluar formulations, derivatives also include chemical derivativeswherein synthetic chemistry is employed to modify a starting materialwhich is a taxane.

Topoisomerase inhibitors, which may be employed in a method of thepresent disclosure include type I topoisomerase inhibitors, type IItopoisomerase inhibitors and type II topoisomerase poisons. Type Iinhibitors include topotecan, irinotecan, indotecan and indimitecan.Type II inhibitors include genistein and ICRF 193 which has thefollowing structure:

Type II poisons include amsacrine, etoposide, etoposide phosphate,teniposide and doxorubicin and fluoroquinolones.

In one embodiment the chemotherapeutic is a PARP inhibitor.

Labels

In one embodiment the payload comprises a fluorescent label, achemi-lluminescent label, a radio label, an enzyme, a dye or a ligand.

A label in accordance with the present disclosure is defined as anymoiety which may be detected using an assay. Non-limiting examples ofreporter molecules include enzymes, radiolabels, haptens, fluorescentlabels, phosphorescent molecules, chemiluminescent molecules,chromophores, photoaffinity molecules, colored particles or ligands,such as biotin.

Label conjugates are generally preferred for use as diagnostic agents.Diagnostic agents generally fall within two classes, those for use in invitro diagnostics, and those for use in vivo diagnostic protocols,generally known as “directed imaging.” Many appropriate imaging agentsare known in the art, as are methods for their attachment to peptidesand polypeptides (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948,and 4,472,509). The imaging moieties used can be paramagnetic ions,radioactive isotopes, fluorochromes, NMR-detectable substances, andX-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰. ¹²⁵I is suitable for use in certain embodiments, andtechnicium^(99m) and/or indium¹¹¹ are particularly suitable due to theirlow energy and suitability for long range detection. Radioactivelylabeled peptides and polypeptides may be produced according towell-known methods in the art. For instance, peptides and polypeptidescan be iodinated by contact with sodium and/or potassium iodide and achemical oxidizing agent such as sodium hypochlorite, or an enzymaticoxidizing agent, such as lactoperoxidase. Peptides may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the peptide.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to peptide arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Fluorescent labels suitable for use as payloads include Alexa 350, Alexa430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G,BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, FluoresceinIsothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, OregonGreen 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red,Renographin, ROX, TAM RA, TET, Tetramethylrhodamine, and/or Texas Red.

Another type of payload is that suitable for use in vitro, is where apeptide is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and avidin andstreptavidin compounds. The use of such labels is well known to those ofskill in the art and is described, for example, in U.S. Pat. Nos.3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149 and4,366,241.

Other methods are known in the art for the attachment for linking apeptide to its conjugate moiety. Some attachment methods involve the useof a metal chelate complex employing, for example, an organic chelatingagent such a diethylenetriaminepentaacetic acid anhydride (DTPA);ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide; and/ortetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody (U.S.Pat. Nos. 4,472,509 and 4,938,948). Peptides or polypeptides may also bereacted with an enzyme in the presence of a coupling agent such asglutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

In one embodiment the label is able to stain or label the nucleus of astem cell.

Viruses Suitable for Use as Payloads in the Present Disclosure

In one embodiment the virus employed in the present disclosure is anenvelope virus, for example selected from a herpesvirus (such as Herpessimplex 1), a poxvirus (such as vaccina virus), a hepadnavirus, aflavivirus, a togavirus, a coronavirus, hepatitis D, orthomyxovirus,paramyxovirus (such as measles or Newcastle disease virus), rhabdovirus,bunyavirus, filovirus, and Rhabdoviridae (such as vesicular stomatitisIndiana virus (VSV).

In one embodiment the virus employed in the present disclosure is anon-envelope virus, for example selected from adenoviridae (such as anadenovirus), papilomaviridae, picornaviridae (such as coxsackie virus orSeneca Valley virus (eg Senecavirus)), reovirus.

In one embodiment the virus is an adenovirus, for example a humanadenovirus, such as selected from a group B virus (in particular Ad3,Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad51 or a chimeria thereof,such as Enadenotucirev), a group C virus (in particular Ad1, 2, 5, 6 ora chimeria thereof), a group D virus (in particular Ad8, Ad10, Ad13,Ad15, Ad17, Ad19, Ad20, Ad22, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39,Ad42, Ad43, Ad44, Ad45, A46, Ad47, Ad48, Ad49, Ad50 or a chimeriathereof), a group E virus (in particular Ad4), a group F virus (inparticular Ad40, Ad41 or a chimeria thereof) and a chimeria of two ormore of group B, C, D, E or F viruses.

The vast majority of viruses have well described proteins associatedwith target cell recognition and uptake. Modification of their tropismto re-direct or enable more selective tumor targeting into oncolyticviruses may be introduced using methods described in rev. in Verheijeand Rottier, Adv. Virology 2012: 798526, 2012.

Additional viral cell surface proteins not involved in native viraltargeting can have targeting motifs engineered onto them (e.g. Ad virionminor coat protein IX Salisch et al., PLoS One 12: e0174728, 2017).

Envelope viruses have an outer membrane (envelope) covering the viruscapsid. The envelope is typically derived from the portions of the hostcell membranes (phospholipids and proteins) but also include some viralproteins. Glycoproteins on the surface of the envelope serve to identifyand bind to receptor sites on the host's membrane. The viral envelopethen fuses with the host's membrane, allowing the capsid and viralgenome to enter and infect the host.

Various oncolytic viruses are disclosed in WO2014/13834, incorporatedherein by reference.

Herpes simplex virus (HSV) enters cells by means of four essentialglycoproteins—gD, gH/gL, gB, activated in a cascade fashion by gDbinding to one of its receptors, nectin1 and HVEM. Retargeting of HSVhas been achieved by the insertion of ligands and scFvs into the gCand/or gD protein or gH (Campadelli-Fiume, G et al., Rev in Med Virol21: 213-226, 2011, Gatta, V PLoS Pathog 11: e1004907, 2015). Oncolyticherpes simplex virus type 1 vectors have been developed for clinicaluse. These viruses are replication competent and have mutations in thegenes that affect viral replication, neuropathogenicity, and immuneevasiveness, and for example include first generation viruses such asNV1020 (R7020), dlsptk, d18.36tk, hrR3, R3616, 1716, second generationviruses such as G207 (MGH-1), 3616UB, SUP, NV1023, third generationviruses such as G47Δ, transcriptional expressing vectors such as G92A,d12.CALP, Myb34.5, transgene expressing vectors such as rRP450, andother viruses such as Talimogene laherparepvec (T-Vec). The HSV-1vectors are the thought to be useful in the treatment of a wide of solidtumors, for example including glioma, melanoma, breast, prostate, colon,ovarian, and pancreatic cancers. The HSV-1 virus infects a broad rangeof cells types and species, it is cytolytic by nature, the replicativelife cycle of the virus results in host cell destruction, it has a wellcharacterised and large genome (152K) but contains many non-essentialgenes providing up to 30K of space for the insertion of therapeuticgenes. Generally, HSV viruses are not mutated in the thymidine kinasegene for safety reasons. Talimogene laherparepvec is an oncolytic herpesvirus, which is approved for use in the treatment of melanoma. Otherherpes bases viruses include G207, SEPREHVIR (HSV-1716), by VirttuBiologics, HSV-1 R3616 mutant, HSV-1 1716 mutant, NV1020 (R7020), R3616mutant (deleted RL1), KM100 mutant has insertions in UL48 (encodes thetransactivator tegument protein pUL48 [VP16]) and RL2 genes, G92A,mutants, Myb34.5 and rQNestin34.5.

Poxvirus—Vaccina virus, such as Modified Vaccinia Ankara (MVA) may beemployed (Galmiche M C et al., J Gen Virol 78: 3019-3027, 1997), MVA maybe replaced with a p14 fusion molecule carrying an inserted scFvdirected against the tumor associate antigen MUC-1 (Paul, S et al.,Viral Immunol 20: 664-671, 2007) See also rev. in Liang L et al.,Viruses 6: 3787-3808, 2014, Hsiao J C et al., J Virol 73: 8750-8761,1999, rev. in Chen T L and Roffler S, Med Res. Rev. 28: 885-928, 2008and Kinoshita T et al., J Biochem 144: 287-294, 2008. JX-594, byJennerex, is a thymidine kinase-deleted Vaccinia virus plus GM-CSF.GL-ONC1 is an attenuated vaccinia virus (Lister strain) that causesregression and elimination of a wide range of solid tumors in preclincalmouse models

Paramyxovirus (such as measles or Newcastle disease virus),

Measles virus (MeV) is a single-stranded, negative-sense, enveloped(non-segmented) RNA virus of the genus Morbillivirus within the familyParamyxoviridae. Measles virus has two envelope glycoproteins: thehemagglutinin (H) attachment protein and the fusion (F) protein.Attachment, entry and subsequent cell-cell fusion is mediated via 2measles receptors, CD46 and the signaling lymphocyte activation molecule(SLAM). See for example rev. in Msaouel P et al., Methods Mol Biol 797:141-162, 2012, Robinson S. and Galanis, E. Expert Opin Biol Ther. 17:353-363, 2017, Aref S et al., Viruses 8. Pii:E294, 2016); (rev. in ChenT L and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita T etal., J Biochem 144: 287-294, 2008), and (Russell S J and Peng K W, CurrTopic Microbiol. Immunol 330: 213-241, 2009, Robinson S and Galanis, EExpert Opin Biol. Ther 17: 353-363, 2017, Aref S et al., Viruses 8. Pii:E294, 2016). Measles virus encoding the human thyroidal sodium iodidesymporter or MV-NIS is an attenuated oncolytic Edmonston (Ed) strain ofmeasles virus. Radioactive Iodine imaging provides a novel technique forNIS gene expression monitoring.

Newcastle disease virus may also be employed.

Adenoviridae Adenoviruses are among the most extensively studied virusesbeing used as oncolytic agents. An array of peptides and proteins havebeen engineered into virion associated viral proteins to alter thenative tropism of the virus (rev. in Verheije M H and Rottier P J M AdvVirol 2012: 798526, 2012). However, all of these are dependent uponviral assembly in the nucleus which presents significant challenges.

Other non-enveloped viruses include Coxsackievirus, Poliovirus andReovirus. See for example rev. in Altan-Bonnet, N, Curr Opin Microbiol32: 77-81, 2016 and Chen Y H et al., Cell 160: 619-630, 2015, rev. inChen T L and Roffler S, Med Res. Rev. 28: 885-928, 2008 and Kinoshita Tet al., J Biochem 144: 287-294, 2008 and rev. in Verheije M H andRottier P J M Adv Virol 2012: 798526, 2012).

There are a numerous adenoviruses for exampleAd5-yCD/mutTKSR39rep-hIL12, such as for the treatment of prostate cancerwas initiated, CGTG-102 (Ad5/3-D24-GMCSF), by Oncos Therapeutics, forexample for the treatment soft tissue sarcomas, Oncorine (H101), CG0070,Enadenotucirev (EnAd) WO2005/118825, OvAd1 and OvAd2 disclosed inWO2008/080003, ONCOS-102, for example for Unresectable Malignant PleuralMesothelioma, and DNX-2401 for example for glioma.

Cavatak is the trade name for a preparation of wild-type CoxsackievirusA21, useful in the treatment of malignant melanoma. Seneca Valley virus(NTX-010) and (SVV-001), for example for small cell lung cancer andneuroblastoma

Reovirus-Reolysin® (pelareorep; Wild-Type Reovirus; Serotype 3 Dearing;Oncolytics Biotech), for example for the treatment of various cancersand cell proliferative disorders.

Vesicular Stomatitis Virus (VSV) VSV is another enveloped virus beingexplored as on oncolytic agent. See for example Betancourt D et al., JVirol 89: 11786-11800, 2015) and rev. in Hastie E and Grdzelishvili V ZJ Gen Virol 93: 2529-2545, 2012).

Proteins Encoded by a Virus

In one embodiment a virus or vector employed in the method of thepresent disclosure comprises a transgene, for example where thetransgene is to replace defective genetic material in the cell, toprovide a new or augmented function in the cell, to sensitize the cellto treatment, to block a function in the cell, or to express atherapeutic protein or peptide.

In one embodiment the virus employed as the payload according to thepresent disclosures, comprises a transgene or transgenes, for exampleencoding an agent independently selected from an RNAi sequence, aprotein, polypeptide or peptide (for example an antibody molecule orbinding fragment thereof, a chemokine, a cytokine, an immunomodulator, afluorescent tag or an enzyme).

This includes but is not limited to unique formats that have shownpreclinical promise but have lacked an effective and economical meansfor delivery e.g. peptides, intrabodies and alternative scaffolds (rev.in Boldicke T, Protein Sci 26: 925-945, 2017, Marschall and Dubel,Comput Struct Biotechnol J 14: 304-308, 2016, Miersch and Sidhu F1000Res5.pii.F1000 Faculty Rev. 1947, 2016, Peptides, Tsomaia Eur J Med Chem94:459-470, 2015, Marschall A L J et al, Mabs 7: 1010-1035, 2015,AlDeghaither D et al., J Clin Pharmacol. 55: S4-S20, 2015))) andincludes agents with therapeutic effects on the tumor cells tumor stemcells, tumor associated endothelium and tumor associated stroma. Ofspecial interest are molecules that could serve multiple functions, forexample as therapeutics, biomarkers and/or diagnostics. The herpessimplex virus thymidine kinase (HSV-TK) gene is a well-establishedpro-drug converting enzyme with a clinically approved pro-drug(ganciclovir—GCV) see for example Holder et al., Cancer Res. 53:3475-3485, 1993, Touraine R L et al., Gene Therapy 5: 1705-1711, 1998),

In addition, the thymidine kinase protein expression can also beexploited to image and track the activity of the virotherapy during thecourse of treatment. Positron emission tomography and single photonemission computed tomography are both methods that are routinely usedfor the detection and monitoring of cancer and cancer therapies and areboth viable means to detect the expression of the thymidine kinaseprotein when an appropriate thymidine kinase substrate is administered(Wang J Q et al., Bioorg Med Chem 13: 549-556, 2005, Tjuvajev J G et al,J Nucl Med 43: 1072-1083, 2002). Alternatively, the NIS gene may be usedand has been explored as an agent for diagnostic and therapeuticpurposes in oncolytic viruses, much like TK (Miller A and Russell SExpert Opin Biol Ther 16: 15-32, 2016, Ravera S et al., Annu Rev Physiol79: 261-289, 2017, Portulano et al., Endocr Rev. 35: 106-149, 2014).

In one embodiment antibodies that interact and inhibit RAS or proteinsin the RAS signaling pathway are encoded in the virus of the presentdisclosure, for example as fusion protein with the GLA-component. RASgenes constitute a multigene family that includes HRAS, NRAS, and KRAS.See for example Bos J L, Cancer Res. 49: 4682-4689, 1989; and Cetin M etal., J Mol Biol. 429:562-573, 2017.

Combination Therapy

In one embodiment the GLA-component is employed in combination with asecond therapy, for example an anti-cancer therapy. This is therapy thatis administered separately to the GLA-component (i.e. is not linked tothe GLA-component).

In one embodiment a combination of chemotherapeutic agents employed is achemotherapeutic described herein, for example a platin and 5-FU or aprodrug thereof, for example cisplatin or oxaplatin and capecitabine orgemcitabine, such as FOLFOX.

In one embodiment the chemotherapy comprises a combination ofchemotherapy agents, in particular cytotoxic chemotherapeutic agents.

In one embodiment the chemotherapy combination comprises a platin, suchas cisplatin and fluorouracil or capecitabine.

In one embodiment the chemotherapy combination in capecitabine andoxaliplatin (Xelox).

In one embodiment the chemotherapy is a combination of folinic acid and5-FU, optionally in combination with oxaliplatin.

In one embodiment the chemotherapy is a combination of folinic acid,5-FU and irinotecan (FOLFIRI), optionally in combination withoxaliplatin (FOLFIRINOX). The regimen consists of: irinotecan (180 mg/m²IV over 90 minutes) concurrently with folinic acid (400 mg/m² [or 2×250mg/m²] IV over 120 minutes); followed by fluorouracil (400-500 mg/m² IVbolus) then fluorouracil (2400-3000 mg/m² intravenous infusion over 46hours). This cycle is typically repeated every two weeks. The dosagesshown above may vary from cycle to cycle.

In one embodiment the chemotherapy combination employs a microtubuleinhibitor, for example vincristine sulphate, epothilone A,N-[2-[(4-Hydroxyphenyl)amino]-3-pyridinyl]-4-methoxybenzenesulfonamide(ABT-751), a taxol derived chemotherapeutic agent, for examplepaclitaxel, abraxane, or docetaxel or a combination thereof.

In one embodiment the combination therapy employs an mTor inhibitor.Examples of mTor inhibitors include: everolimus (RAD001), WYE-354,KU-0063794, papamycin (Sirolimus), Temsirolimus, Deforolimus(MK-8669),AZD8055 and BEZ235(NVP-BEZ235).

In one embodiment the combination therapy employs a MEK inhibitor.Examples of MEK inhibitors include: AS703026, CI-1040 (PD184352),AZD6244 (Selumetinib), PD318088, PD0325901, AZD8330, PD98059,U0126-EtOH, BIX 02189 or BIX 02188.

In one embodiment the combination therapy employs an AKT inhibitor.Examples of AKT inhibitors include: MK-2206 and AT7867.

In one embodiment the combination employs an aurora kinase inhibitor.Examples of aurora kinase inhibitors include: Aurora A Inhibitor I,VX-680, AZD1152-HQPA (Barasertib), SNS-314 Mesylate, PHA-680632,ZM-447439, CCT129202 and Hesperadin.

In one embodiment the combination therapy employs a p38 inhibitor, forexample as disclosed in WO2010/038086, such asN-[4-({4-[3-(3-tert-Butyl-1-p-tolyl-1H-pyrazol-5-yl)ureido]naphthalen-1-yloxy}methyl) pyridin-2-yl]-2-methoxyacetamide.

In one embodiment the combination employs a Bcl-2 inhibitor. Examples ofBcl-2 inhibitors include: obatoclax mesylate, ABT-737,ABT-263(navitoclax) and TW-37.

In one embodiment the combination therapy comprises a checkpointinhibitor, for an anti-PD-1 inhibitor or an anti-PD-L1 inhibitor.

In one embodiment the chemotherapy combination comprises anantimetabolite such as capecitabine (xeloda), fludarabine phosphate,fludarabine (fludara), decitabine, raltitrexed (tomudex), gemcitabinehydrochloride and cladribine.

In one embodiment the chemotherapy combination comprises ganciclovir,which may assist in controlling immune responses and/or tumourvasculation.

In one embodiment the chemotherapy includes a PARP inhibitor.

In one embodiment the combination therapy includes an inhibitor ofcancer metabolism with specific inhibition of the activity of the DHODHenzyme.

In one embodiment one or more therapies employed in the method hereinare metronomic, that is a continuous or frequent treatment with lowdoses of anticancer drugs, often given concomitant with other methods oftherapy.

In one embodiment, there is provided the use of multiple cycles oftreatment (such as chemotherapy) for example 2, 3, 4, 5, 6, 7, 8.

In one embodiment the chemotherapy is employed in a 28 day cycle.

In one embodiment the molecules of the present disclosure are providedin a pharmaceutical composition comprising a excipient, diluent and/orcarrier. In one embodiment the composition is as a parenteralformulation.

Parenteral formulation means a formulation designed not to be deliveredthrough the GI tract. Typical parenteral delivery routes includeinjection, implantation or infusion.

In one embodiment the parenteral formulation is in the form of aninjection. Injection includes intravenous, subcutaneous, intra-cranial,intrathecal, intra-tumoural or intramuscular injection. Injection asemployed herein means the insertion of liquid into the body via asyringe.

In one embodiment the parenteral formulation is in the form of aninfusion.

Infusion as employed herein means the administration of fluids at aslower rate by drip, infusion pump, syringe driver or equivalent device.In one embodiment, the infusion is administered over a period in therange of 1.5 minutes to 120 minutes, such as about 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 15, 16, 17, 18, 19 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 65, 80, 85, 90, 95, 100, 105, 110 or 115 minutes.

In one embodiment, the formulation is for intravenous (i.v.)administration. This route is particularly effective because it allowsrapid access to the majority of the organs and tissue and is particularuseful for the treatment of metastases, for example establishedmetastases especially those located in highly vascularised regions suchas the liver and lungs.

Therapeutic formulations typically will be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other parenteral formulationsuitable for administration to a human and may be formulated as apre-filled device such as a syringe or vial, particular as a singledose.

As discussed above the formulation will generally comprise apharmaceutically acceptable diluent or carrier, for example a non-toxic,isotonic carrier that is compatible with the virus, and in which thevirus is stable for the requisite period of time.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a dispersant or surfactant such as lecithin or a non-ionicsurfactant such as polysorbate 80 or 40. In dispersions the maintenanceof the required particle size may be assisted by the presence of asurfactant. Examples of isotonic agents include sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.

In one embodiment there is provided a kit of parts comprising aGLA-component according to the present disclosure and a payload, whereinthe payload is linked or unlinked to said GLA-component.

“Comprising” in the context of the present specification is intended tomean “including”.

Where technically appropriate, embodiments of the invention may becombined.

Embodiments are described herein as comprising certainfeatures/elements. The disclosure also extends to separate embodimentsconsisting or consisting essentially of said features/elements.

Technical references such as patents and applications are incorporatedherein by reference.

The technical backgrounds is part of the technical disclosure of thepresent specification and may be used as basis for amendments becausethe discussion therein is not limited to discussing the prior art as italso includes a discussion of the technical problems encountered in thefield and the application of the present technology.

Any embodiments specifically and explicitly recited herein may form thebasis of a disclaimer either alone or in combination with one or morefurther embodiments.

The present application claims priority from U.S. Ser. Nos. 62/554,530,62/569,403, 62/554,533, 62/569,411, 62/584,565 and 62/593,014. Each ofthese applications are incorporated by reference. These applications maybe employed as the basis for a correction to the present specification.

The invention will now be described with reference to the followingexamples, which are merely illustrative and should not in any way beconstrued as limiting the scope of the present invention.

EXAMPLES

FIG. 1A-D Shows various representations of GLA protein structures.

FIG. 1E Shows an embodiment of a GLA-component according to the presentdisclosure.

FIG. 2 Shows Protein S (PrS) and annexin staining of breast cancer celllines treated with peroxide to induce apoptosis. A, human MDA-231 cellstreated with peroxide and stained with FITC-PrS. B, untreated MDA-231cells stained as in A. C, treated MDA-231 cells stained with annexin. D,human MCF-7 cells treated with peroxide and stained with PrS. E, murineMET-1 cells, as in D. F, murine 4T1 cells, as in D.

FIG. 3 Shows overlapping, yet distinct, cellular localization of PrS andannexin. A, murine 4T1 cells treated with peroxide and stained with Cy5PrS (RED) and FITC annexin (GREEN). Light arrow, co-localized signals;red arrows, cells staining with PrS and not annexin; green arrow, cellstaining relatively brighter with annexin but less bright with PrS,indicating distinct binding patterns (insets show PrS and annexinstaining separately). B, treated 4T1 cells stained with FITC PrS and Cy5annexin. Green arrows, cells staining with PrS and not annexin. C, Cy5annexin staining of treated 4T1 cells pre-incubated with 1,000-foldexcess of cold annexin.

FIG. 4 Shows staining of apoptotic COS-1 cells with PrS and annexin.Cells were treated with t-BHP as described and stained with FITC annexin(left) and Cy5 PrS (right). Arrows indicate subcellular structurespresumed to be apoptotic bodies.

FIG. 5 Shows differential staining of extracellular vesicles with PrSand annexin. Extracellular vesicles were prepared from 4T1 cells andstained with FITC PrS (GREED) and Cy5 annexin (RED). Arrows indicatevesicles staining with annexin only (RED arrow), PrS only (GREEN arrow)and both proteins (light arrow).

FIG. 6 Shows subcellular localization of PrS and annexin. A, B,apoptotic 4T1 cells were stained with FITC PrS (GREEN arrows) and Cy5annexin (RED arrows); light arrows, co-localization. C, Possibleapoptotic bodies.

FIG. 7 Shows internalization of PrS within 5 minutes. Apoptotic 4T1cells were stained with FITC PrS (green) and Cy5 annexin (red) andimaged within 10 min of the addition of the proteins. A, Merged image.B, Hoescht nuclear stain alone.

FIG. 8 Shows BLI images of 4T1 tumors in mice.

FIG. 9 SPECT imaging of effect of doxorubicin on 4T1 tumors, usingradiolabeled PrS and annexin. Mice with 4T1 breast cancer tumors wereimaged with 99mTc PrS (A and B), or annexin (C and D), before (A and C)and 24 h after doxorubicin (B and D).

FIG. 10 Shows SPECT imaging of cyclohexamide-treated mice. Five mice perpanel are shown before (A and C) and 24 h after (B and D) treatment. Themice were imaged with either ^(99m)Tc PrS (A and B), or annexin (C andD), Arrows indicate increased liver signal.

FIG. 11 Shows localization of Cy5 PrS to infected spleen. CD1 mice wereinfected with bioluminescent Listeria and imaged on day 2 postinfection. The mice were injected with Cy5 PrS 30 min before sacrifice,and the spleens removed and frozen. Modestly infected (A) and controluninfected (C) mice are shown. Sections of the infected (B) anduninfected (D) spleens of each mouse in the Cy5 channel are shown,merged with phase contrast images.

FIG. 12 Shows localization of Cy5 PrS to tumors treated withdoxorubicin. Mice implanted with 4T1 breast cancer tumors were treatedwith doxorubicin (right panels) or left untreated (left panels). 24hours later the mice were injected intravenously with Cy5 PrS andsacrificed 30 min later. The tumors were removed, frozen, and sectionedfor fluorescence microscopy. Merged Cy5/phase contrast images from fourdifferent mice are shown.

FIG. 13 Shows differentiation of TSCs. TSCs were cultured in thepresence (left) or absence (right) of growth factors. Arrows in theright panel indicate giant cells characteristic of differentiation.

FIG. 14 Shows PrS staining of trophoblast stem cells and differentiatedtrophoblasts. Trophoblast stem cells (left) were differentiated intotrophoblast giant cells (right) by withdrawal of growth factors. Thecells were stained with Cy5 PrS and imaged.

FIG. 15 Shows MSC differentiation. MSC were treated as described in thetext, for differentiation into adipocytes (upper panels) or osteoblasts(lower panels). Differentiated cells exhibited the expected morphologyin each case.

FIG. 16 Shows MSCs stained with PrS (green), annexin (red), and Hoechst(blue). Cells were imaged within 10 min of addition of the stainmixture.

FIG. 17 Shows TSCs stained with PrS (green, lightest area), annexin(red, light around the cell membrane), and Hoechst (blue). Cells wereimaged within 5 min of addition of the stain mixture.

FIG. 18 Shows differential staining of TSC vesicles. TSCs were stainedas in FIG. 17. The group of cells are secreting large vesicles thatstain with annexin (red) and not PrS (green).

FIG. 19 Shows PrS staining of C17.2 neural progenitor cells. The cellswere stained with PrS-FITC and imaged with standard (non-confocal)microscopy.

FIG. 20 Shows internalization of PrS into TSC at 4C. FITC PrS (green)and Cy5 annexin (red) were added to TSC at 4C and imaged with confocalmicroscopy.

FIG. 21 Shows lineage-negative, SCA-1/c-kit staining cells from mousebone marrow. The cells were not stained with either PI (propidiumiodide; to detect dead cells) or PrS at this point in the analysis.Absence of staining for hematopoietic lineages (left panel) and stainingof c-kit and SCA1 (right panel) defines the population of HSC, shown ingreen (lightest areas).

FIG. 22 PrS staining of long-term HSC. HSC were isolated as in FIG. 1,and stained with FITC PrS. SLAM pattern was determined with Cy7(x-axis).

FIG. 23 PrS staining of short-term HSC. HSC were isolated as in FIG. 1,and stained with FITC PrS. SLAM pattern was determined with Cy7(x-axis).

FIG. 24 Shows internalization of PrS in long-term HSC. HSC were preparedas described, stained for PrS, and examined with confocal microscopy.Green (lightest areas), FITC PrS; blue, Hoescht nuclear stain; red, PI.Note that PI stain is excluded from the nucleus, indicating the cellsare alive.

FIG. 25 Shows an example of dead HSC exhibiting nuclear PI.

FIG. 26 GLA-mediated delivery is non-toxic to cells

This specification also includes sequences 1 to 6, in the associatedsequence listing.

This project initiated the testing of labeled recombinant PrS as an invivo imaging agent for SPECT (Single Photon Computed Tomography).Surprisingly it was found that the molecule rapidly internalized intoapoptotic cells. This unexpected finding led us to explore thephenomenon further, whereupon we found that PrS was also internalizedinto a subset of non-apoptotic stem cells of several types.

PrS is protein S GLA domain and protein S EGF domain as shown in SEQ IDNO: 6.

Methods

For fluorescence, conjugation of Cy5 and FITC was achieved usingAmersham (GE Heathcare) and Molecular Probes (Invitrogen) labeling kits,respectively, according to the instructions of the manufacturers. Bothkits provide columns for the removal of unconjugated fluorophore.Initially, 0.77 mg of PrS (Fraction 2) in 1 ml and 0.77 mg of annexin in1 ml were labeled with FITC to test for specificity of binding toapoptotic cells. For co-localization and competition studies 0.68 mg ofPrS (Fraction 3) in 1 ml and 0.68 mg of annexin were labeled with Cy5.For confocal microscopy, 0.76 mg of PrS from the second shipment waslabeled with FITC and the previously labeled Cy5-conjugated annexin wasused. It should be noted that the precise efficiency of labeling was notdetermined and the recovery from the columns was assumed to be 85%,according to the instructions of the manufacturers of the labeling kits.Thus, the relative staining intensity of the two proteins in any casemay reflect these contingencies. The cells were stained for 30 mininitially, but it was subsequently determined that less than 5 min wassufficient. To test PrS for apoptotic cell-specificity, four breastcancer cell lines were initially employed; human MDA-231 and MCF7 andmurine 4T1 and MET-1. Subsequently, COS-1 monkey kidney cells were alsoused. Apoptosis was induced with hydrogen peroxide or tertiary-Butylhydroperoxide (t-BHP). The cells were plated in 24-well plates at 6×10⁴cells per well or Eppendorf chamber slides at 1×10⁴ cells per well, andapoptosis was induced the next day, using 2 mM H₂O₂, or t-BHP for timepoints from 30 min to 2 hrs. After induction, the wells were washed withAnnexin Binding Buffer (AB; Santa Cruz Biotech), and stained withlabeled protein. From past experience and the literature, 5.5 μg/ml ofannexin protein was used for staining. This amount was adjusted forequimolar addition of PrS by assuming the molecular weights of annexinto be 36 kD and the recombinant PrS to be 30 kD, based on the gel imagesprovided. The cells were stained for 15 min. Hoechst 33342 dye was usedfor visualizing nucleic acid. The wells were then washed with AB andobserved using the EVOS fluorescence microscope while still viable. Forconfocal microscopy, the Leica SP8 microscope in the Stanford CellSciences Imaging Facility was employed. The wells were then washed withAB and observed using the Leica sp8 microscope. Hoechst 33342 dye wasused for visualizing nuclei. For toxicity studies, PrS was added totrophoblast stem cells (TSCs) and the viability tested with trypan blueusing a Nexcelom Cellometer.

To test the labeled proteins for the ability to detect tumors, 5×10⁴ 4T1-luc cells were implanted into groups of 5 male BALB/c mice, in theleft axillary fat pad. The mice were imaged with in vivo bioluminescenceimaging (BLI) each day to monitor tumor growth, starting at 1 week postimplantation. The mice were then treated on day 11 post implantationwith 13 mg/kg body weight of intraperitoneal (IP) doxorubicin, and BLIwas performed the next day. Control mice bearing tumors were leftuntreated with doxorubicin. 48 hrs post treatment the mice were imaged 1hr after intravenous tracer injection (anesthesia 1.3 g/kg of urethaneIP), with single head A-SPECT gamma camera (Gamma Medica); 1 mm pin holecollimator, 128 steps into a 128×128 imaging matrix, 15 seconds perstep, 2.7 cm ROR; FOV=upper chest/neck. The injected dose of eachprotiein was 160 μl (800 μCO. The animals were then sacrificed andbiodistribution was performed. For the cyclohexamide treatmentexperiment, groups of 5 young (7 week old) male Swiss Webster mice wereanesthetized (1.3 g/kg of urethane IP) and injected intravenously with50 mg/kg cycloheximide. 1 hr 45 min after cycloheximide injection,tracer was injected (PrS=180 ul/1.2 mCi per dose; annexin V =170 μl/1.05mCi per dose). 45 min after tracer injection, the mice were imaged with10 min static whole body images using a single head parallel holecollimator (128×128 matrix) on the A-SPECT gamma camera.

To test for the specific localization of fluorescent PrS to apoptoticsites due to infection in live animals, CD1 mice were injectedintravenously with bioluminescent Listeria monocytogenes. This bacterialpathogen infects many organs including the spleen, in which extensiveapoptosis of monocytes and granulocytes occurs. At certain times postinfection, spleen is the primary site of bacterial replication and sosplenic BLI signals from the bacteria can be correlated with thelocalization of probes for apoptosis. Mice were infected and imaged eachday. When splenic signals were evident (day 2 post infection for 2×10⁵colony forming units of bacteria in 8 week old CD1 female mice), 300mg/kg body mass of Cy5 PrS was injected into mice, the animals weresacrificed 30 min later, and the spleens removed, frozen in OCT, andsectioned for fluorescence microscopy. Uninfected control mice wereemployed.

Flow cytometry was performed. Freshly labeled FITC PrS, prepared asdescribed above, was employed. Murine hematopoietic stem cells (HSCs)are routinely purified in this laboratory. The cells were isolated fromnormal mouse bone marrow by staining for c-Kit+, lineage-negative cells.To further characterize the cells, SLAM marker staining was alsoperformed. These markers stain cells that self-renew and differentiate,whereas non-staining HSCs can only differentiate. Subsequent stainingwith FITC PrS revealed the percent positive in SLAM-staining cells, asshown in the Results. The cells were then sorted for FITC and examinedwith confocal microscopy, using Hoechst 33342 for nuclear visualization.

Results

To assess PrS binding specificity in the context of apoptosis in cellculture, we employed several human and murine breast cancer cell lines.Apoptosis was induced with peroxide as described above, and FITC PrSbinding was assessed. Examples of these experiments are shown in FIG. 2.Untreated cells exhibited minimal binding, such as shown in panel B ofFIG. 2. Concentrations of peroxide and incubation times were chosen suchthat only a minority of cells would be affected, because at higherconcentrations and/or longer incubation times the cells detached andstaining and microscopy was not possible. In addition, the presence ofmany unaffected cells served as an internal negative control within eachfield. FITC-annexin showed specificity for apoptosis similar to PrS,serving as an internal positive control. We then tested the two proteinsfor co-localization and competitive binding. For co-localization, bothFITC and Cy5 labeled PrS and annexin were prepared. 4T1 cells weretreated with peroxide and stained with Cy5 and FITC labeled PrS andannexin, using both combinations of fluorophores. The cells were thenvisualized in the EVOS fluorescence microscope. The results are shown inFIG. 3. Under the conditions tested, all the brightly staining cellsexhibited staining with both proteins. However, whether using Cy5 orFITC, PrS appeared to stain some cells that annexin did not, albeitweakly (FIG. 3). The relative staining intensity of different cells byeach protein sometimes differed between the two probes, i.e., sometimesannexin stained two cells with equal intensity and PrS did not, and viceversa (FIG. 3A, green arrow and insert). Thus, while both probesgenerally stained the same cells, they appeared to exhibit subtledifferences. In the competition assay, increasing excess amounts ofunlabeled annexin were pre-incubated with apoptotic 4T1 cells for 15 minand the cells were then stained with Cy5 PrS. Surprisingly, the stainingof PrS was not blocked by even 1,000 fold excess of annexin, the highestexcess amount tested (FIG. 3C), although these proteins are thought tobind to the same target molecule, exposed PS. Co-staining of annexin andPrS was observed with many cell types. While the two proteins generallystained the same cells in each cell type, other differences becameapparent. In particular, some objects smaller than cells weredifferentially stained (FIG. 4). These objects, which were present inincreased numbers after peroxide treatment, were interpreted asapoptotic bodies; membrane-bound cell fragments produced during thefragmentation of apoptotic cells. As shown in FIG. 4, PrS stained theseentities, whereas annexin did not, although some of these objects didstain with both proteins. This observation was unexpected. To furtherexplore the differential staining of sub cellular entities,extracellular vesicles (EVs) were prepared from 4T1 murine tumor cellsusing a standard centrifugation protocol. The two proteins alsodifferentially stained these vesicles (FIG. 5), a result that may havebiological and therapeutic implications.

EVs, specifically exosomes, microvesicles (MVs) and apoptotic bodies(ABs), are presumed to play key roles in cell-cell communication viatransfer of biomolecules between cells. The biogenesis of these types ofEVs differs, and they originate from either the endosomal (exosomes) orplasma membranes (MV) or are products of programmed cell death (ABs).All mammalian cells are thought to secrete EVs. Each type of EV cantransfer molecular cargo to both neighboring and distant cells,affecting cellular behaviors such as those involved in tumor developmentand progression. In fact, EVs may play a role in nearly all thehallmarks of cancer, including sustaining proliferative signaling,evading growth suppression, resisting cell death, reprogramming energymetabolism, acquiring genomic instability, and developing the tumormicroenvironment. They have also been implicated in the induction ofangiogenesis, control of invasion, initiation of premetastatic niches,sustaining inflammation, and evading immune surveillance. Immune cellsappear to also communicate through EVs and my recognize EVs as signalsfrom tumor cells, infected tissues and wounds. A deeper understanding ofthe biology of EVs and their contribution to the hallmarks of cancer isleading to new possibilities for diagnosis and treatment of cancer.Development of additional EV surface markers is essential to advancingthis field and PrS may be such a determinant.

Following these studies with fluorescence microscopy, the subcellularlocalization of the staining by PrS and annexin was then evaluated viaconfocal microscopy. Murine 4T1 cells (lacking the Luc-GFP reporters)were plated on 8-part chamber slides at 1×10⁴ cells per chamber andapoptosis was induced with 2 mM H₂O₂ or t-BHP (2 hr exposure) the nextday. The cells were then washed and stained for 15 min with PrS andannexin. Hoechst 33342 dye was used to stain nucleic acid. In all cases,the most brightly staining cells were stained with both probes. However,in many cells labeled PrS was observed in the cytoplasm, whereas thelabeled annexin was not (FIG. 6). Although annexin was internalized andappears in vesicles of a few cells, internalized annexin together withsurface localized PrS in the same cell was not observed. These resultswere unexpected, because the two proteins are both presumed to bind PS.To further study the internalization of PrS, a time course experimentwas performed. Apoptotic 4T1 cells were stained for 5 min with Cy5annexin and FITC PrS, and observed within 5 min of the addition of theprobes. PrS was observed in the cytoplasm of these cells immediately,indicating internalization within 5 min (FIG. 7). The time course imagesalso showed that PrS and annexin did not always stain the same cellsequally at early time points. The cells in FIG. 7 appear to be indifferent stages of apoptosis, as the cell on the left shows anuncondensed nucleus surrounded by an apparently intact nuclear membrane,whereas the right cell exhibits the strong staining often characteristicof chromatin condensation that occurs later in the apoptotic process.Staining patterns such as these may indicate that PrS binds earlier inapoptosis than annexin. Although purely conjecture at this point, such apreference would explain many of the differences between these proteinsthat have been observed so far. For example, the staining of some cellsby PrS and not annexin, such as in FIGS. 3A and B may be due to PrSbinding earlier in the process of apoptosis. To examine PrS localizationin live animals, several experiments were performed. These studiesemployed chemical and infectious induction of apoptosis in vivo, as wellas the localization of PrS to tumors treated with doxorubicin, which isknown to induce apoptosis. SPECT imaging using HYNIC-labeled PrS andannexin was performed in animals given 4T1luc breast tumors and treatedwith doxorubicin. Because the 4T1 tumors have been labeled withluciferase, they can be imaged in mice using in vivo bioluminescenceimaging (BLI). One of the images from this experiment is shown in FIG.8. This method can be used to evaluate tumor implantation and to followprogression in individual animals over time. ⁹⁹mTc labeled PrS andannexin were then employed for SPECT imaging of animals treated withdoxorubicin and controls. An example of the results is shown in FIG. 9.The images of the head and thorax of the two animals show non-specificaccumulation of the PrS probe in the salivary gland, and a low signal tonoise ratio using this probe. Therefore, the threshold of the display inthe PrS images shown was lowered to reveal more background, resulting inthe brighter false-color of the images. The low signal-to-noise ratio islikely due to HYNIC labeling of only 1 mg of protein, which issub-optimal, and also due to the inability to perform controlled studiesof HYNIC:protein labeling ratio.

SPECT imaging of mice treated with cyclohexamide, which inducesapoptosis in the liver, was also performed (FIG. 10). In FIG. 10, thewhole-body images of 5 mice are shown in each panel. As with manyradiolabeled probes, background is seen in the kidneys. Treatment of themice with cyclohexamide increased the annexin SPECT signal in the liver.Again, the PrS showed low signal compared to annexin. Annexin was ableto detect the apoptotic livers of cyclohexamide treated mice, whereasPrS showed only slight increase of signal in the liver due to treatment.To test the localization of PrS to apoptotic tissues and treated tumorsindependently of SPECT imaging and the concomitant complications ofHYNIC labeling, mice infected with bacteria that induce apoptoticresponses and tumor bearing mice were injected with Cy5 PrS. Forinfection, we employed Listeria monocytogenes, a bacterial pathogenlabeled with luciferase and well characterized for BLI. CharacteristicBLI signals from the spleen provide for excellent co-localizationstudies. CD1 mice were infected as described above and were imaged withBLI on day 2 post infection. The mice were then injected with Cy5 PrSand 30 min later sacrificed, and the spleens removed for sectioning andfluorescence microscopy (FIG. 11). In all cases, splenic sections frominfected mice showed much greater Cy5 fluorescence signals thancontrols. In FIG. 11, the infected mouse shown displayed low photoncounts, indicating the infection had not yet progressed very far in thisanimal. Many mice exhibit 10 times this signal intensity from the spleenon this day. However, the Cy5 channel fluorescence was still very strongrelative to the uninfected control shown. This result may reflect theongoing innate immune response to infection, as granulocytes andmacrophages have been shown to be the main source of annexin signal insuch animals (these cells are programmed for apoptosis to limit tissuedestruction).

The localization of fluorescent PrS to 4T1 tumors treated withdoxorubicin was then tested. Mice implanted with tumors were treatedwith doxorubicin as described above and Cy5 PrS was injectedintravenously 30 min prior to sacrifice and removal of the tumors forsectioning and fluorescence microscopy. The results are shown in FIG.12. Areas of intense staining were observed in the treated animals,whereas more modest signal was observed from the untreated tumorsections. Although some untreated tumors did exhibit small areas ofhigher signal than background, no signals of similar intensity to thetreated tumors were observed in any of the untreated sections.

Stem cells are distinct in phenotype from differentiated cells and mayexpress PS non-apoptotically to avoid the induction of immune responses.Trophoblast stem cells (TSCs) differentiate into several types oftrophoblasts in culture. TSCs are prepared from mouse uterine scrapingsgrown in the presence of fibroblast growth factor, activin, and heparin.TSCs spontaneously differentiate into giant cells when these factors areremoved from the medium (FIG. 13). TSCs stained with PrS, whereasdifferentiated trophoblasts derived from these cells in culture did notstain (FIG. 14). We have also determined that PrS is internalized intostem cells without apoptotic induction. This result confirmsobservations made in tumor cell lines, in which apoptosis was induced.Without induction of apoptosis, minimal staining was observed in tumorcells. To test for internalization in stem cells, we employedmesenchymal stem cells (MSCs) and TSCs. MSCs were prepared from mousebone marrow. The bone marrow was flushed from mice and cultured for 6days in the absence of growth factors. During this incubation, MSCs andhematopoietic stem cells (HSCs) replicate, whereas fibroblasts adherebut do not multiply beyond a few generations. After 6 days, a monolayeris visible. Upon passage by trypsinization, the adherent MSCs areretained, whereas the HSCs, which grow in suspension, are lost. Thefibroblasts do not persist due to absence of growth factors and are alsonot retained. Thus, this simple procedure results in a nearlyhomogeneous population of MSCs. To confirm the identity of these cells,we treated the cultures separately with dexamethasone and glycerolphosphate (to induce differentiation into osteoblasts) or dexamethasoneand indomethacin (to induce differentiation into adipocytes). Theresults are shown in FIG. 15. In response to the above treatments,differentiated cells showed the appearance of the respective cells.Adipocytes contained large fat vesicles and osteoblasts were dark withdistinctive intracellular collagen and mineralization.

To assess subcellular staining pattern, undifferentiated MSC werestained with PrS and annexin, as well as Hoechst nuclear stainingreagent, and observed with confocal microscopy. Results of theobservations are shown in FIG. 16. PrS was rapidly internalized. In thecase of MSC, about 1 in 20 cells stained with PrS, consistent withprevious data, however the precise percentage that stained was notdetermined. The morphology of MSCs is heterogeneous, and the cellssecrete abundant material into the medium, some of which adheres to thesurface of the chamber slide, making resulting in background in some ofthe images. Nonetheless, the data clearly show internalized PrS, within5 minutes of addition and annexin on the surface. TSCs were also stainedand imaged as was done with the MSCs. The observations confirminternalization into these cells as well, which also occurs within 5minutes of addition of the protein. The results are shown in FIG. 17.TSCs are morphologically quite variable, and can be multinucleate in theabsence of differentiation, as can be seen in the figure. As with theMSCs, these primary cells shed abundant material into the medium, someof which we have established as extracellular vesicles (previous data).This material again makes the imaging difficult. Some EVs stain withannexin and not PrS, and this phenomenon can be seen in TSCs, in FIG.18. In this image of a cluster of TSCs, vesicles being released by thecells stain with annexin and not PrS, which is internalized. Thesepatterns raise interesting questions regarding the specificity andbinding targets of PrS and annexin. The two proteins are both reputed tobind PS. However, the differential binding to EVs as well as distinctsubcellular localization patterns suggest that they are not binding inexactly the same manner. Further studies will be required to establishthe basis of this distinction, which may prove to be significant. Wehave also observed PrS staining of the neural progenitor cell line C17.2(FIG. 19), which is a transformed cell line capable of differentiationin vitro into astrocytes and other neuronal cells. Approximately 5% ofthese transformed cells stained, although this percentage is anestimate. Remarkably, entry into TSCs occurred even when the cells werechilled to 4° C. (FIG. 20). However, it must be noted that the chambercould not be continually chilled once placed on the microscope.Nevertheless, the temperature could not have risen much within the 5 mintime frame of the imaging procedure. This result, while provocative,must clearly be repeated under more controlled conditions. Should thefinding be substantiated, the mechanism would have to be veryinteresting indeed.

We have succeeded in staining hematopoietic stem cells (HSC) with PrS.Using flow cytometry we determined that HSC stain with PrS, and haveobserved internalization of PrS in these cells with confocal microscopy.HSC were identified and isolated using fluorescence activated cellsorting (FACS). The cells were identified in bone marrow aslineage-negative, SCA/c-kit positive cells (FIG. 21). These were thenstained with FITC-PrS. Two populations of HSC, short-term and long-term,can be identified with the pattern of SLAM marker staining. The SLAM(Signaling Lymphocyte Activation Molecule) markers CD48, CD150, CD229and CD244 differentially stain HSC with distinct patterns such that SLAMpattern-positive staining is indicative of the ability to bothself-renew and differentiate, whereas SLAM pattern-negative HSC can onlydifferentiate. PrS stained a subset of long-term HSC (FIG. 22), and alsoshort-term HSC (FIG. 23). The cells shown are propidium iodide(PO-negative, meaning that they are all live cells. This result confirmsprevious experiments demonstrating that a subset of stem cells stainswith PrS without the induction of apoptosis.

We then proceeded to test for internalization of PrS into HSC. Thisexperiment was complicated by many factors. Perhaps the most difficultwas the survival in culture of HSC, which die in large numbers in mediumovernight. We therefore had to time the experiment such that flowcytometry analysis and confocal microscopy occurred on the same day.Furthermore, the cells are not adherent, making microscopy less thanoptimal. To make microscopy more efficient, the cells were resuspendedin a small drop of medium. Finally, we needed to make sure that thePrS-stained cells analyzed by microscopy were still alive. Many HSC diedduring the processes of analysis and isolation. Therefore, PI was addedand scanned in addition to the Hoescht nuclear stain, and anotherchannel was employed. The presence of PI-bright nuclei indicated deadcells. Despite these difficulties and the complexities of timing, wewere able to perform the experiment, and confirmed internalization ofPrS into live HSC (FIG. 24). The cells were confirmed as alive by lackof nuclear PI staining. However, some cells were dead or dying as shownin FIG. 25. Despite the complexity and length of the experiment shown,the results show internalization.

Finally, in FIG. 26, we have performed preliminary toxicity studies onTSC, and determined that at a concentration of 135 μg/ml, viability wasreduced only by a very minimal extent after 30 min, from 78% to 74%,relative to PBS. Considering that, at this level, 10% of the culturevolume was PrS-containing solution, this result confirmed ourqualitative observations that PrS is basically non-toxic to stem cells,and the minor toxicity observed could well be due to contaminatingcontents of the preparation itself. Lower concentrations of PrS showedno effect on viability. The highest level of protein tested was morethan 1000 times the concentration used for staining. While full toxicitystudies, which were not formally part of this project, will require muchmore extensive tests, in our hands PrS exhibits very little toxicity.

Summary

The above results have shown that PrS is rapidly internalized into anarray of cells expressing PrS, including stem cells of many types, whichsuggests that PrS possesses unique characteristics amenable tomanipulation toward the goal of developing a therapeutic agent. Inaddition, the difference in specificity between PrS and annexin such asseen in FIGS. 3 and 7 suggests that binding itself is different betweenthese two proteins. The mere fact that annexin is a tetramer and PrS isa monomer cannot explain these differences and these data suggest thatsome other component on the cell surface may be involved in PrS binding.The mechanism of binding, specificity, and internalization of PrS, aswell as the capability of modular manipulation provide a host ofpossibilities.

Example 2

Stem cells are distinct in phenotype from differentiated cells and mayexpress PS non-apoptotically to avoid the induction of immune responses.Stem cells were stained with a GLA domain molecule of the presentdisclosure comprising a payload of a fluorescent label, without theinduction of apoptosis.

Trophoblast stem cells, (FIG. 14) which differentiate into several typesof trophoblasts in the placenta, stained with Protein S, whereasdifferentiated trophoblasts derived from these cells in culture did notstain. The stain was able to distinguish between in vivo differentiatedstems cells and cells differentiated in vitro.

This data the molecules of the present disclosure may be employed totarget cells in vivo or in ex vivo samples.

REFERENCES

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1. A method of targeting a stem cell said method comprising the step ofcontacting cells with a molecule comprising a payload linked to agamma-carboxyglutamic acid component (GLA-component) wherein saidGLA-component comprises a GLA domain or an active fragment thereof, andsaid molecule does not comprise an active catalytic domain from a GLAprotein.
 2. A method according to claim 1, wherein GLA domain or activefragment thereof is independently selected from thrombin, factor VII,factor IX, factor X, protein C, protein S, protein Z, osteocalcin,matrix GLA protein (MGP), GAS6, transthyretin (TTR),inter-alpha-trypsin-inhibitor, periostin, proline rich gla 1 (PRRG1),proline rich gla 2 (PRRG2), proline rich gla 3(PRRG3), and proline richgla 4 (PRRG4).
 3. A method according to claim 2, wherein the GLA domainor active fragment thereof is from protein S.
 4. A method according toclaim 1, wherein the GLA-component further comprises an EGF domain, forexample a calcium binding EGF domain.
 5. A method according to claim 1,wherein the construct comprises an EGF domain selected from thrombin,factor VII, factor IX, factor X, protein C, protein S, protein Z,Osteocalcin, Matrix GLA protein, GAS6, Transthretin, Periostin, Prolinerich GLA 1, Proline rich GLA 2, Proline rich GLA 3 and Proline rich GLA4.
 6. A method according to claim 5, wherein the EGF domain is fromprotein S.
 7. A method according to claim 1, wherein the GLA-domaincomponent further comprises a Kringle domain.
 8. A method according toclaim 7, wherein the Kringle domain is from a protein selected from thegroup comprising Activating transcription factor 2 (ATF); Factor XII(F12); thrombin (F2); Hyaluronan-binding protein 2 (HABP2); Hepatocytegrowth factor (HGF); Hepatocyte growth factor activator (HGFAC); Kremenprotein 1 (KREMEN1); KREMEN2; Lipoprotein(a) (LPA); LPAL2;Macrophage-stimulating protein (MSP or MST1);Phosphoinositide-3-kinase-interacting protein 1 (PIK3IP1); Tissueplasminogen activator (PLAT); Urokinase (PLAU); Plasmin (PLG); PRSS12;Tyrosine-protein kinase transmembrane receptor ROR1 (ROR1); andTyrosine-protein kinase transmembrane receptor ROR2 (ROR2).
 9. A methodaccording to claim 1, wherein the GLA-component comprises a sequenceshown in SEQ ID NO:
 6. 10. A method according to claim 1, wherein themethod is performed in vitro.
 11. A method according to claim 1, whereinthe delivery is to a cell in vivo, for example wherein the moleculecomprising the GLA component and the payload are administered to apatient.
 12. A method according to claim 1, wherein the molecule targetsthe exterior of the stem cell.
 13. A method according to claim 1,wherein the molecule is internalised in the stem cell.
 14. A methodaccording to claim 1, wherein the cell is non-apoptotic.
 15. A methodaccording to claim 1, wherein the cell is apoptotic, for example adiseased stem cell.
 16. A method according to claim 1, wherein the stemcell is an embryonic stem cell or an adult stems cells includingprogenitor cells, and haemotopoietic stem cells, myogenic stem cells,osteoprogenitor stem cells, neural stem cells, mesenchymal stem cell,such as satellite cells, radial glial cells, bone marrow stromal cells,periosteum, pancreatic progenitor cells, endothelial progenitor cells,blast cells and trophoblast stem cells.
 17. A method according to claim1, wherein the stem cell expresses a surface marker CD34.
 18. A methodaccording to claim 1, wherein the stem cell is negative for lineagepositive surface markers (i.e. is Lin −ve).
 19. A method according toclaim 1, wherein the stem cell is CD90+ve, CD133+ve, CD105+ve, CD45+,Lin−ve, CD48−ve, and CD244−ve.
 20. A method according to claim 1,wherein the stem cell is Lin−ve, CD34+ve, CD38−ve, CD45RA−ve, CD90+veand CD49f_+ve.
 21. A method according to claim 1, wherein the stem cellis non-cancerous.
 22. A method according to claim 1, wherein the stemcell is a cancer stem cell.
 23. A method according to claim 22, whereinthe cancer stem cell is of epithelial origin.
 24. A method according toclaim 22, wherein cancer stem cell expresses a surface marker selectedfrom CD44 (which is over expressed in at least breast, ovary, prostate,pancreatic, squamous, and bladder cancer), CD133 (which is overexpressed in at least brain, colon, lung, prostate cancer andmedulloblastoma), CD24, CD90, CD271, CD4f, CD13 and combinations of twoor more of the same.
 25. A method according to claim 1, wherein the stemcell is of haemotopoietic origin.